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

RESEARCH ARTICLE Identification of positively selected genes in human pathogenic treponemes: Syphilis-, yaws-, and bejel-causing strains differ in sets of genes showing adaptive evolution Denisa Maděra ´nkova ´ 1 , Lenka Mikalova ´ 2 , Michal Strouhal 2 , S ˇimon Vadja ´k 1 , Ivana Kuklova ´ 3 , Petra Pospı ´s ˇilova ´ 2 , Lenka Krbkova ´ID 4 , Pavlı ´na Kos ˇčova ´ 1 , Ivo Provaznı ´k 1 , David S ˇmajsID 2 * 1Department of Biomedical Engineering, Brno University of Technology, Brno, Czech Republic, 2Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic, 3Department of Dermatology, 1st Faculty of Medicine, Charles University in Prague, Prague, Czech Republic, 4Department of Children’s Infectious Diseases, Faculty of Medicine and University Hospital, Masaryk University, Brno, Czech Republic *[email protected]ni.cz Abstract 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. PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007463 June 19, 2019 1 / 18 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Maděra ´nkova ´D, Mikalova ´L, Strouhal M, Vadja ´k S ˇ, Kuklova ´I, Pospı ´s ˇilova ´P, et al. (2019) Identification of positively selected genes in human pathogenic treponemes: Syphilis-, yaws-, and bejel-causing strains differ in sets of genes showing adaptive evolution. PLoS Negl Trop Dis 13 (6): e0007463. https://doi.org/10.1371/journal. pntd.0007463 Editor: Ana LTO Nascimento, Instituto Butantan, BRAZIL Received: August 29, 2018 Accepted: May 14, 2019 Published: June 19, 2019 Copyright: ©2019 Maděra ´nkova ´et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: This work was supported by grants from the Grant Agency of the Czech Republic (1725455S; 18-23521J) to D.S and (GP14-29596P) to M.S. and by funds from the Faculty of Medicine, Masaryk University to junior researchers Lenka Mikalova ´-Pas ˇtěkova ´and Michal Strouhal. The 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. Author summary In the genus Treponema there are several human and animal pathogens that include the causative agent of syphilis (Treponema pallidum ssp. pallidum; TPA), the causative agent of yaws (T.p. ssp. pertenue; TPE), and the causative agent of endemic syphilis (T.p. ssp. endemicum; TEN). T.paraluisleporidarum causes venereal spirochetosis in rabbits. We used whole genome sequences of 11 treponemal strains together with additional 62 draft genomic data to identify genes evolving under positive selection. The identified genes evolving under positive selection partly overlapped with the genes previously reported as recombinant and were found to be different in treponemal subspecies. Since both genetic recombination and positive selection could allow a survival of pathogenic bacteria despite the human immune response, identification of such genes could predict the major antigens recognized by the human immune system and also identify the most suitable components for development of an anti-treponemal vaccine. Introduction Adaptive evolution including positive selection plays crucial roles in the evolution of bacterial human pathogens and both have been well documented on a genome-wide scale in a number of bacterial genera including Escherichia,Helicobacter,Neisseria,Listeria,Salmonella,Streptococcus,Campylobacter, and Actinobacillus [1–8]. Pathogenic treponemes are both human and animal pathogens. Human pathogens include Treponema pallidum ssp. pallidum (TPA), the causative agent of syphilis, T.p. ssp. pertenue (TPE, the causative agent of yaws), and T.p. ssp. endemicum (TEN, the causative agent of bejel) while animal pathogens include TPE causing non-human primate infections [9–12], and T.paraluisleporidarum ecovar Cuniculus (TPeC; formerly denoted as Treponema paraluiscuniculi), the causative agent of venereal spirochetosis in rabbits, and T.paraluisleporidarum ecovar Lepus, which infects hares [13–15]. Although the recent work of Edmondson et al. [16] reported successful long-term cultivation of T.pallidum in a tissue culture system, most of the data on treponemal genetics comes from the whole genome sequencing studies [17,18]. The above-listed pathogens are monomorphic, i.e., highly similar at the genetic level and all the genomes of these pathogenic treponemes characterized to date share a genetic identity of 98% or higher [17,18]. The group of human pathogens (TPA, TPE, TEN) are even more Positive selection in treponemes PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007463 June 19, 2019 2 / 18 funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. related sharing a genetic identity over 99.7% [17,19,20]. Currently, 14 complete treponemal genomes have been published including six TPA genomes, six TPE genomes, one TEN genome, and one TPeC genome [12,19–29]. In addition, 23, 25, 8, and 6 draft whole genome sequences of treponemal strains or isolates were published recently by Arora et al. [30], Pinto et al. [31], Sun et al. [32], and Marks et al. [33], respectively. The genomes of pathogenic treponemes related to T.pallidum contain no prophages or insertion sequence-elements [21,34], or plasmids [35]. Therefore, recombination is expected to be quite infrequent among these treponemes due to a lack of mobile genetic elements [21]. However, traces of both intragenomic DNA recombinations via gene conversion [36–38] and intergenomic homologous recombination after DNA horizontal gene transfer have been described [20,25]. In addition, traces of positive selection have been detected in previously published papers including TPA and TPE comparisons [19], comparisons within TPA strains [39], and detailed intrastrain analysis [40]. However, no comprehensive analysis of positively selected loci in the genomes of pathogenic treponemes has been performed to date. Despite the fact that treponemes related to Treponema pallidum are monomorphic bacteria with extremely low level of genetic diversity [18], it has been shown that human immunity does not protect against different subspecies and not even against different syphilis strains [41]. Therefore, divergent genes encoding differences in proteomes of individual treponemal strains and subspecies are likely of importance for development of syphilis vaccine. In this communication, the whole genome sequences of 11 treponemal strains were systematically analyzed for the presence of positive selection. The identified genes were further reanalyzed relative to all sequences available in 62 draft genomes published to date. The causative agents of syphilis, yaws, and bejel differed in sets of positively selected genes. Moreover, several synthetic peptides covering positively selected protein regions were found to interact with syphilitic sera. Materials and methods Strains used in this study A set of 11 treponemal genomic sequences was examined in this study and included genomes of five TPA strains (Nichols, DAL-1, Mexico A, SS14, Chicago B), four TPE strains (CDC-2, Gauthier, Samoa D, Fribourg-Blanc), one TEN strain (Bosnia A), and one strain of TPeC (Cuniculi A). An overview of the complete genome sequences used is shown in Table 1. Subsequent analysis of selected genes was performed on additional 62 draft genomes (Arora et al. [30]; Pinto et al. [31]; Sun et al. [32]; GenBank genome TPA sequences [UW074B, UW189B, UW228B, UW254B, UW391B] and TEN Iraq B [CP032303.1]). The overall algorithm is shown in Fig 1. Identification of genes under adaptive evolution The TPE Samoa D was used as a reference genome, and all 1068 orthologous genes were extracted from the other 10 complete genomes using given annotation coordinates. The orthologous sequences from the complete genomes were aligned at the codon level using Matlab R2013a software and the Bioinformatics Toolbox. Only genes where at least three nonsynonymous mutations at different sites occurred were further analyzed with respect to the presence of positive selection. A BLAST search was used to determine orthologous sequences in the draft genomes. These sequences were aligned in Matlab at the nucleotide level because large number of ambiguous sites precluded proper automatic ORF localization. Then the sequences from the draft genomes were scanned for nucleotide differences. After filtering sites with unknown nucleotides, insertions, and deletions, only orthologs with more than five nucleotide Positive selection in treponemes PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007463 June 19, 2019 3 / 18 differences at different sites were analyzed further. These orthologs were aligned with the corresponding genes from the complete genomes to determine the ORF and identify the number of nonsynonymous mutations. At the same time, the TPeC orthologs were excluded in this step due to frequent sequential diversity and due to the lack of pathogenicity of TPeC to humans. The removal of the TPeC orthologs did not change the number of detected positively selected genes. Compared to an analysis of whole genomes, no new locus with more than three nonsynonymous mutations at different sites was identified during the analysis of draft genomes. Table 1. Treponemal genomes analyzed in this study. TP strain�Place and year of isolation Reference GenBank Accession number, Genome sequence reference TPA Nichols Washington, D.C., USA; 1912 [42] CP004010.2, [21,26] TPA DAL-1 Dallas, USA; 1991 [43] CP003115.1, [29] TPA SS14 Atlanta, USA; 1977 [44] CP004011.1, [24,26] TPA Mexico A Mexico City, Mexico; 1953 [41] CP003064.1, [25] TPA Chicago Chicago; 1951 [41] CP001752, [22] TPE CDC-2 Akorabo, Ghana; 1980 [45] CP002375.1, [19] TPE Gauthier Brazzaville, Congo; 1960 [46] CP002376.1, [19] TPE Samoa D Apia, Samoa; 1953 [41] CP002374.1, [19] TPE Fribourg-Blanc Guinea; 1966 [9,10] CP003902.1, [12] TEN Bosnia A Bosnia; 1950 [47] CP007548, [20] TPeC Cuniculi A unknown; before 1957 [41] CP002103.1, [27] �Additional genome sequences of TPE Ghana-051 and CDC 2575 became available recently [28] and TPA Sea81-4 was published as a whole genome sequence [23]. https://doi.org/10.1371/journal.pntd.0007463.t001 Fig 1. The algorithm used for identification of positively selected genes. The original search for positively selected genes started with identification of gene orthologs with 3 or more nucleotide differences leading to nonsynonymous amino acid replacements. The original search was performed on a set of 11 complete treponemal genomes listed in Table 1. Subsequently, orthologous gene sequences extracted from published treponemal draft genomes were used and the Cuniculi A orthologs were removed due to frequent sequential diversity and due to lack of pathogenicity of TPeC to humans. Orthologs from draft genomes were used when available and positively selected genes were analyzed within treponemal subspecies using branch-site PAML model analysis. https://doi.org/10.1371/journal.pntd.0007463.g001 Positive selection in treponemes PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007463 June 19, 2019 4 / 18 For each analyzed gene from the complete genomes, a maximum likelihood phylogenetic tree with 50 bootstrap replicates was constructed using MEGA 6 [48]. Different evolution models (Kimura 2-parameter [49], Tamura 3-parameter [50], and Tamura-Nei [51]) were applied to each gene. The trees of each gene were compared by calculating Robinson-Foulds distances [52] using R software (packages phytools and phangorn) [53]. The comparison showed that the choice of the evolution model did not significantly change the topology of the tree; the Tamura-Nei evolution model was chosen for the construction of all phylogenetic trees. A calculation of mutational rate ratio ωbetween two gene sequences was the basis for the positive selection analysis. The ωwas calculated as a ratio of nonsynonymous to synonymous mutational rates. The ratio indicates negative purifying selection (0 <ω<1), neutral evolution (ω= 1), and positive selection (ω>1) [54]. A set of selected genes from complete genomes was tested relative to positive selection using the maximum likelihood method using the CODEML of the PAML software package [55]. PAML version 4 [56] and its user interface PAMLX [57] were used in our study. For each analyzed gene, its maximum likelihood phylogenetic tree was used as an input tree. The CODEML offers several different codon evolutionary models, and the statistical likelihood ratio test (LRT) was used to compare the codon evolutionary model to the null model. The Bayes empirical Bayes method (BEB) [58] was then used to evaluate the posterior probability of sites considered to have been positively selected. The CODEML models could produce different results (i.e., a list of sites under positive selection) since they calculate different parameter estimates. Site models allow ωto vary in each site (codon) within the gene. Statistical testing was required for sites with ω>1. Two pairs of models were predominantly used since their LRTs have low false-positive rates. M1a (nearly neutral evolution) was compared to M2a (positive selection) [58,59] and M7 (beta) was compared to M8 (beta & ω) [60]. Our preliminary testing found that the two model pairs gave the same or very similar results. Therefore we chose to use the M7-M8 model pair. The M7 model is a null model that allows 10 classes of sites with a ωbeta-distribution within the interval 0 �ω�1. Sites with ω>1 are not allowed. The alternative M8 model adds an eleventh class of sites with ω>1. Each site was tested to determine the class to which it belongs. The LRT compares twice the log-likelihood difference 2Δl= 2(l 1 -l 0 ) between the M7 model (log likelihood value l 0 ) and the M8 model (log likelihood value l 1 ) to the χ 2 distribution [61]. If the twice log-likelihood difference is above a critical χ 2 value, then the null model is rejected, and the positive selection is statistically significant. A considerable disadvantage of the site models is that ωwas calculated as an average over all codons of the site. Therefore, the site models are not suitable for the data where ωalso varies between lineages. In contrast, the branch-site models search for positive selection in sites and pre-specifies lineages where different rates of ωmay occur [62]. Sequences of lineages are a priori divided into a group of foreground lineages where positive selection may occur and group of background lineages where only purifying selection or neutral evolution occurs. We used branch-site model A, which allows four classes of sites and different setups of foreground lineages to be tested depending on the gene phylogeny. In branch-site model A, all lineages under purifying selection with a low value of ω 0 belong to site class 0. Weak purifying selection and neutral evolution with ω 1 near to value 1 are allowed in site class 1. In site class 2a, a proportion of class 0 sites in foreground lineages is under positive selection with ω 2 >1. Similarly, site class 2b is a proportion of class 1 sites under positive selection with ω 2 >1. The null model for LRT has ω 2 = 1. Critical values of LRT (2Δl) are 2.71 at 5% and 5.41 at 1% [63]. The posterior probabilities of suggested sites under positive selection were calculated using the BEB method. The average pairwise p-distances (APD) and average number of mutations (transitions and transversions), calculated using MEGA-X [64], were used to evaluate genetic diversity. A Positive selection in treponemes PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007463 June 19, 2019 5 / 18 pairwise deletion of sites with gaps/missing data was used. The Fisher exact statistical test was used to assess the significance of the changes between average numbers of mutations. Synthetic peptides and chemiluminescent detection of serological reactions Synthetic biotinylated peptides, covering protein regions containing positively selected residues, were designed. Peptide synthesis was performed by JPT Peptide Technologies (Berlin, Germany) on a 50–200 nmol scale. The lyophilized peptides were resuspended in TBS buffer (25 mM Tris, 150 mM NaCl, pH = 7.2) at 1 mM concentrations and were stored at −20˚C. Prior to further use, synthetic peptides were diluted 1000x in TBS buffer. Streptavidin-coated 96 well plates (Pierce Streptavidin Coated High Binding Capacity White 96-Well Plates; Thermo Scientific, Rockford, USA) were washed three times with 200 μl of washing buffer (TBS buffer containing Tween 20 (0.05%) and Bovine Serum Albumin, BSA (0.1%) (Sigma-Aldrich, Prague, Czech Republic); then 100 μl of diluted peptide was added to each well and incubated for 30 min at room temperature as recommended by the manufacturer (Thermo Scientific) with mild shaking. Subsequently, each well was washed three times with washing buffer (200 μl in each step); then 100 μl of blocking buffer SuperBlock Blocking Buffer in TBS (Thermo Scientific) were added and incubated for 30 min at room temperature with mild shaking. Each well was washed three times with washing buffer (200 μl in each step) and 100 μl of diluted sera (1:500 in washing buffer) were added and incubated for 30 min at room temperature with mild shaking. Each well was washed three times with washing buffer (200 μl in each step) and 100 μl of diluted secondary antibody conjugated with horseradish peroxidase were subsequently added (1:2000 in washing buffer; Goat Anti-Human IgG/IgA/ IgM Horseradish Peroxidase Conjugate; Life Technologies, Carlsbad, USA) and incubated for 30 min at room temperature with mild shaking. Each well was then washed three times with washing buffer (200 μl in each step); then 100 μl of chemiluminescent detection solution (Super Signal ELISA Pico Chemiluminescent Substrate; Thermo Scientific) was added. Luminescence was measured on a TriStar 2 LB 942 luminometer with a Modular Multimode Microplate Reader (Berthold Technologies, Bad Wildbad, Germany). Each experiment was performed at least three times. A signal was considered positive when it was higher than the average of the three lowest values for each serum plus five standard deviations of the average value. Ethics statement The human sera were collected from adult patients diagnosed with syphilis at the Department of Dermatology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic. Sera from child patients diagnosed with Lyme disease were obtained from the Department of Children’s Infectious Diseases, Faculty of Medicine and University Hospital, Masaryk University, Brno, Czech Republic. All clinical samples were obtained after patients or parents of involved children signed an informed consent. The design of the study was approved by the ethics committee of the Faculty of Medicine, Masaryk University. All human sera were collected under established guidelines. Results Identification of positively selected genes A comparison of 47 orthologous gene sequences from the complete genomes where at least three nonsynonymous mutations at different sites occurred was used to identify positively selected genes using the site and branch-site models of the CODEML in PAML package [55]. Positive selection in treponemes PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007463 June 19, 2019 6 / 18 The completely sequenced genomes are listed in Table 1 and include 11 genomes. In addition, 25 draft TPA genomes [31], 23 draft TPA and TPE genomes [30], 8 TPA genomes [32], 5 TPA genomes from GenBank (UW074B, UW189B, UW228B, UW254B, UW391B), and one TEN (Iraq B) were also analyzed. The overall algorithm is shown in Fig 1. In all cases of complete treponemal genomes, the genome structure was identical or very similar allowing straightforward identification of gene orthologs. However, in many cases, draft genome sequences were either incomplete or contained many ambiguous bases precluding their use in analyses. This resulted in a variable number of sequences used for identification of positively selected sites within individual loci. Altogether, 22 positively selected genes were identified in the TP genomes using site model analysis in PAML (Table 2). The number of positively selected amino acid sites varied from 1 to 65, with a median value of 8.5. A list of positively selected protein sites identified using PAML software (site and branch-site models) as well as PAML-identified positively selected protein sites within treponemal subspecies are shown in S1 Table. Average pairwise p-distances (APD) were calculated for each of the 22 genes from 10 complete genomes (Cuniculi A genome was removed from analysis) and from the draft genomes. The APD value for each gene was compared with APD w10 = 0.000525 of the 10 complete genomes without 54 variable loci (listed in S2 Table) which can be considered as a background level of polymorphism. All 22 genes evolving under adaptive evolution had elevated nucleotide substitution density. Out of these 22 genes, 14 genes were previously reported as recombinant (Table 2). These genes are listed in Table 3. Functionally, these genes comprised the tpr genes (tprC,D,G,I,J, L), outer membrane proteins (TP0133, TP0136, TP0548, TP0856, TP0858, TP0865), and genes encoding the outer membrane biogenesis protein (TP0326 [BamA]) and methyl-accepting chemotaxis protein (TP0488 [Mcp-2]). Putative recombination loci were most frequently identified in TEN strain (n = 9) while in other treponemes there were fewer predicted recombinant loci (TPA, n = 7; TPeC, n = 1; TPE, n = 1). When recombinant sequences were removed from analyses, positive selection among 14 these loci (Table 3) was found mostly within TPA strains or isolates (n = 9), within TPE (n = 4), between TEN and TPA/TPE sequences (n = 2), between TPA and TPE strains (n = 1) and between TPA and TPE/TEN sequences (n = 1). Positively selected genes with no recombination events described so far A list of the 8 positively selected genes is shown in Table 4. These genes include tprF gene, genes encoding outer membrane proteins (TP0515, TP0733, TP0859), subtilisin-like proteins (TP0314, TP0462), enzymes (TP0619), and hypothetical protein (TP0126b). Evidence of positive selection from analyses performed within the strains belonging to different treponemal subspecies and analyses from the PAML branch-site model revealed that positive selection was found mostly between TPA and TPE strains (n = 6), and within TPA strains or isolates (n = 2). In one case (TP0859), positive selection was found both between TPA and TPE strains (n = 1), and between TEN and TPA/TPE (n = 1). Identified positively selected genes in various treponemal species and subspecies are shown in Table 5 and Fig 2. While Table 5 lists all identified recombinant and positively selected genes in TPA, TPE, and TEN groups of strains or isolates, Fig 2 shows only recombinant or positively selected genes that were identified within a particular subspecies. Distribution of genetic diversity among TPA, TPE, and TEN strains To test whether genetic diversity in treponemal genomes was distributed evenly along the chromosome, an average pairwise p-distance (APD) and average number of mutations (ANM, Positive selection in treponemes PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007463 June 19, 2019 7 / 18 transitions and transversions) were calculated (Table 6) for concatenation of 54 genes (S2 Table) representing a total length of 86.8 kbp (7.6% of the genome length). These 54 genes included the tpr genes, genes with traces of possible recombination events (according to Gray et al. [38], and Čejkova ´et al. [19]), genes showing inter-strain variability between TPE and TPA strains and their paralogs (according to Čejkova ´et al. [19]), genes showing intra-strain variability [40], and previously identified positively selected genes (according to Čejkova ´et al. [19]) (S2 Table). The TPA strains contained greater genetic diversity within these genes compared to TPE strains (APD (TPA) = 420.0�10 −5 versus APD (TPE) = 384.2�10 −5 ). Note that the average pairwise p-distances within TPA strains (compared to TPE/TEN strains) are lower when complete genomes were analyzed but higher within selected 54 loci (Table 6). In addition, genetic distance within TPA strains (compared to TPE/TEN strains) is markedly lower in complete genomes without selected 54 genes compared to whole genome analyses (p = 0.0008). Table 2. A set of 22 genes evolving under adaptive evolution that was identified using site and branch-site model analysis in the PAML program. Gene Gene name Protein No. of positively selected protein sites identified by PAML site or branch-site model (no. of analyzed sequences) Previously published evidence of recombination References Gene average pairwise pdistances TP0117 tprC Tpr protein C 22 (41) + [38] 0.009171 TP0126b hypothetical protein 2 (69) - 0.005899 TP0131 tprD Tpr protein D 65 (41) + [38] 0.033553 TP0133 outer membrane protein a 3 (66) + [20] 0.004718 TP0136 fibronectin binding protein 5 (54) +[65] 0.016599 TP0314 subtilisin-like protein a 7 (49) - 0.026378 TP0316 tprF Tpr protein F 5 (33) - 0.003250 TP0317 tprG Tpr protein G 8 (39) + [38] 0.003241 TP0326 bamA BamA 7 (64) + [20,25,66] 0.002131 TP0462 lipoprotein, subtilisinlike protein a 44 (60) - 0.009186 TP0488 mcp2 methyl-accepting chemotaxis protein 50 (64) + [20,25,67] 0.002691 TP0515 outer membrane protein 15 (66) - 0.001381 TP0548 FadL-like protein b 24 (52) + [67] 0.010881 TP0619 Fe, Mn superoxide dismutase a 7 (40) - 0.029166 TP0620 tprI Tpr protein I 14 (38) + [38] 0.006651 TP0621 tprJ Tpr protein J 52 (40) + [38] 0.023909 TP0733 OprG/OmpW-like ion-channel b 1 (62) - 0.005125 TP0856 FadL-like protein b 16 (62) + [68] 0.003663 TP0858 FadL-like protein b 2 (61) + [33,68] 0.005894 TP0859 FadL-like protein b 7 (33) - 0.004191 TP0865 FadL-like protein b 13 (64) + [30] 0.004340 TP1031 tprL Tpr protein L 9 (58) + [20] 0.007283 a protein predictions by Naqvi et al. [69] b protein predictions by Radolf and Kumar [70] https://doi.org/10.1371/journal.pntd.0007463.t002 Positive selection in treponemes PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007463 June 19, 2019 8 / 18 Serological reactivity of human syphilis and Lyme disease sera with synthetic biotinylated peptides derived from positively selected proteins Synthetic biotinylated peptides were designed to cover protein regions where positively selected amino acid residues were detected. These peptides were tested for reactivity with the patient’s syphilis sera (Table 7). As a control, serum from a patient with Lyme disease was used. A positive result was obtained for one to seven peptides (out of 8 tested) depending on the serum used. At the same time, serum obtained from Lyme disease patient failed to Table 3. A set of 14 positively selected genes with previously detected recombination events. Gene Gene name Protein Putative recombination in Evidence of positive selection among non-recombinant sequences TP0117 tprC Tpr protein C TPA, TEN within TPA between TPA and TPE TP0131 tprD Tpr protein D two alternative tprD and tprD2 alleles a no TP0133 outer membrane protein b TEN between TEN/TPE and TPA TP0136 fibronectin binding protein TPA within TPA TP0317 tprG Tpr protein G TPA within TPA TP0326 bamA BamA TPA, TEN within TPA TP0488 mcp2 methyl-accepting chemotaxis protein TPA, TEN within TPA TP0548 FadL-like protein c TEN within TPA within TPE TP0620 tprI Tpr protein I TPE within TPE TP0621 tprJ Tpr protein J TPA, TPeC within TPA TP0856 FadL-like protein c TEN between TEN and TPA/TPE TP0858 FadL-like protein c TEN within TPE TP0865 FadL-like protein c TPA, TEN within TPA within TPE between TEN and TPA/TPE TP1031 tprL Tpr protein L TEN within TPA a tprD and tprD2 alleles existed in both TPA and TPE strains [18,71] b protein predictions by Naqvi et al. [69] c protein predictions by Radolf and Kumar [70] https://doi.org/10.1371/journal.pntd.0007463.t003 Table 4. Positively selected genes revealed by the PAML program with no recombination events described so far. Gene Gene name Protein Positively selected branch TP0126b hypothetical protein between TPA and TPE TP0314 subtilisin-like protein a between TPA and TPE TP0316 tprF Tpr protein F between TPA and TPE TP0462 lipoprotein, subtilisin-like protein a within TPA TP0515 outer membrane protein within TPA TP0619 Fe, Mn superoxide dismutase a between TPA and TPE TP0733 OprG/OmpW-like ion channel b between TPA and TPE TP0859 FadL-like protein b between TPA and TPE between TEN and TPA/TPE a protein predictions by Naqvi et al. [69] b protein predictions by Radolf and Kumar [70] https://doi.org/10.1371/journal.pntd.0007463.t004 Positive selection in treponemes PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007463 June 19, 2019 9 / 18 associated with genome decay. 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