Arturo Centurion-Lara

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.

The ability of Treponema pallidum, the syphilis spirochete to colonize various tissues requires the presence of surface-exposed adhesins that have been difficult to identify due to the inability to culture and genetically manipulate T. pallidum. Using a Borrelia burgdorferi-based heterologous system and gain-in-function approach, we show for the first time that a highly immunogenic lipoprotein TP0435 can be differentially processed into multiple isoforms with one variant stochastically displayed on the spirochete surface. TP0435 was previously believed to be exclusively located in T. pallidum periplasm. Furthermore, non-adherent B. burgdorferi strain expressing TP0435 acquires the ability to bind to a variety of host cells including placental cells and exhibits slow opsonophagocytosis in vitro similar to poor ex vivo phagocytosis of T. pallidum by host macrophages reported previously. This phenomenon of production of both surface and periplasmic immunogenic lipoprotein isoforms has possible implications in immune evasion of the obligate pathogen T. pallidum during infection.

An effective mechanism for introduction of phenotypic diversity within a bacterial population exploits changes in the length of repetitive DNA elements located within gene promoters. This phenomenon, known as phase variation, causes rapid activation or silencing of gene expression and fosters bacterial adaptation to new or changing environments. Phase variation often occurs in surface-exposed proteins, and in Treponema pallidum subsp. pallidum , the syphilis agent, it was reported to affect transcription of three putative outer membrane protein (OMP)-encoding genes. When the T. pallidum subsp. pallidum Nichols strain genome was initially annotated, the TP0126 open reading frame was predicted to include a poly(G) tract and did not appear to have a predicted signal sequence that might suggest the possibility of its being an OMP. Here we show that the initial annotation was incorrect, that this poly(G) is instead located within the TP0126 promoter, and that it varies in length in vivo during experimental syphilis. Additionally, we show that TP0126 transcription is affected by changes in the poly(G) length consistent with regulation by phase variation. In silico analysis of the TP0126 open reading frame based on the experimentally identified transcriptional start site shortens this hypothetical protein by 69 amino acids, reveals a predicted cleavable signal peptide, and suggests structural homology with the OmpW family of porins. Circular dichroism of recombinant TP0126 supports structural homology to OmpW. Together with the evidence that TP0126 is fully conserved among T. pallidum subspecies and strains, these data suggest an important role for TP0126 in T. pallidum biology and syphilis pathogenesis.

Pathogens adapt and evolve in response to pressures exerted by host environments, leading to generation of genetically diverse variants. Treponema pallidum subspecies pallidum displays a substantial amount of interstrain diversity. These variants have been identified in various parts of the world, indicating transmission linkage between geographical regions. Genotyping is based on molecular characterisation of various loci in the syphilis treponeme genome, but still require further development and continued research, as new bacterial types are continually being detected. The goal for studying the molecular epidemiology of Treponema pallidum variants is the global monitoring of the transmission of genetically distinct organisms with different drug sensitivities and, potentially, different virulence proprieties.

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. 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. 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. Uncultivable treponemes represent bacterial species and subspecies that are obligate pathogens of humans and animals causing diseases with distinct clinical manifestations. Treponema pallidum subsp. pallidum causes sexually transmitted syphilis, a multistage disease characterized in humans by localized, disseminated, and chronic forms of infection, whereas Treponema pallidum subsp. pertenue (agent of yaws) and Treponema pallidum subsp. endemicum (agent of bejel) cause milder, non-venereally transmitted diseases affecting skin, bones and joints. The genetic basis of the pathogenesis and evolution of these microorganisms are still unknown. In this study, a high quality whole genome sequence of the T. pallidum subsp. endemicum Bosnia A strain was obtained using a combination of next-generation sequencing approaches and compared to the genomes of available uncultivable pathogenic treponemes. Relative to all known genomes of Treponema pallidum subspecies, no major genome rearrangements were found in the Bosnia A. The Bosnia A strain clustered with other yaws-causing strains, while syphilis-causing strains clustered separately. In general, the Bosnia A genome showed similar genetic characteristics to yaws treponemes but also contained several sequences thought to be unique to syphilis-causing strains. This finding suggests a possible role of repeated horizontal gene transfer between treponemal subspecies in shaping the Bosnia A genome.

Using the rabbit model of syphilis, the Sea81-4 strain of Treponema pallidum subsp. pallidum has been found to be more likely than other strains to invade the central nervous system (CNS). To identify possible explanations for this important phenotype at the genomic level, we sequenced the Sea81-4 strain genome.

The pathogenic non-cultivable treponemes include three subspecies of Treponema pallidum (pallidum, pertenue, endemicum), T. carateum, T. paraluiscuniculi, and the unclassified Fribourg-Blanc treponeme (Simian isolate). These treponemes are morphologically indistinguishable and antigenically and genetically highly similar, yet cross-immunity is variable or non-existent. Although all of these organisms cause chronic, multistage skin and systemic disease, they have historically been classified by mode of transmission, clinical presentations and host ranges. Whole genome studies underscore the high degree of sequence identity among species, subspecies and strains, pinpointing a limited number of genomic regions for variation. Many of these “hot spots” include members of the tpr gene family, composed of 12 paralogs encoding candidate virulence factors. We hypothesize that the distinct clinical presentations, host specificity, and variable cross-immunity might reside on virulence factors such as the tpr genes. Sequence analysis of 11 tpr loci (excluding tprK) from 12 strains demonstrated an impressive heterogeneity, including SNPs, indels, chimeric genes, truncated gene products and large deletions. Comparative analyses of sequences and 3D models of predicted proteins in Subfamily I highlight the striking co-localization of discrete variable regions with predicted surface-exposed loops. A hallmark of Subfamily II is the presence of chimeric genes in the tprG and J loci. Diversity in Subfamily III is limited to tprA and tprL. An impressive sequence variability was found in tpr sequences among the Treponema isolates examined in this study, with most of the variation being consistent within subspecies or species, or between syphilis vs. non-syphilis strains. Variability was seen in the pallidum subspecies, which can be divided into 5 genogroups. These findings support a genetic basis for the classification of these organisms into their respective subspecies and species. Future functional studies will determine whether the identified genetic differences relate to cross-immunity, clinical differences, or host ranges. Pathogenic treponemes include three subspecies of Treponema pallidum (pallidum, pertenue, endemicum), T. carateum, T. paraluiscuniculi, and the unclassified Fribourg-Blanc treponeme. Although they share morphology and have very similar antigenic profiles, they have traditionally been distinguished by mode of transmission, host specificity and the clinical manifestations that they cause. The molecular basis for these disease characteristics is not known. Comparative genomics has revealed that sequences differences among the species and subspecies are found in very localized regions of the chromosome. Many of these regions of sequence variation are found in the tpr genes, which encode a family of twelve candidate virulence factors, many of which are predicted to be outer membrane proteins. Most of the tpr-specific sequence changes are consistent within subspecies or species, supporting the historical classification of these organisms into separate subspecies and species. Functional studies are needed to determine whether any of the tpr gene differences are related to differences in host range, immunity, or clinical manifestations.

Although the threeTreponema pallidumsubspecies (T. pallidumsubsp.pallidum,T. pallidumsubsp.pertenue, andT. pallidumsubsp.endemicum),Treponema paraluiscuniculi, and the unclassified Fribourg-Blanc treponeme cause clinically distinct diseases, these pathogens are genetically and antigenically highly related and are able to cause persistent infection. Recent evidence suggests that the putative surface-exposed variable antigen TprK plays an important role in both treponemal immune evasion and persistence.tprKheterogeneity is generated by nonreciprocal gene conversion between thetprKexpression site and donor sites. Although each of the above-mentioned species and subspecies has a functionaltprKantigenic variation system, it is still unclear why the level of expression and the rate at whichtprKdiversifies during infection can differ significantly among isolates. To identify genomic differences that might affect the generation and expression of TprK variants among these pathogens, we performed comparative sequence analysis of the donor sites, as well as thetprKexpression sites, among eightT. pallidumsubsp.pallidumisolates (Nichols Gen, Nichols Sea, Chicago, Sea81-4, Dal-1, Street14, UW104, and UW126), threeT. pallidumsubsp.pertenueisolates (Gauthier, CDC2, and Samoa D), oneT. pallidumsubsp.endemicumisolate (Iraq B), the unclassified Fribourg-Blanc isolate, and the Cuniculi A strain ofT. paraluiscuniculi. Synteny and sequence conservation, as well as deletions and insertions, were found in the regions harboring the donor sites. These data suggest that thetprKrecombination system is harbored within dynamic genomic regions and that genomic differences might be an important key to explain discrepancies in generation and expression oftprKvariants among theseTreponemaisolates.

In the rabbit model of syphilis, infection phenotypes associated with the Nichols and Chicago strains of Treponema pallidum (T. pallidum), though similar, are not identical. Between these strains, significant differences are found in expression of, and antibody responses to some candidate virulence factors, suggesting the existence of functional genetic differences between isolates. The Chicago strain genome was therefore sequenced and compared to the Nichols genome, available since 1998. Initial comparative analysis suggested the presence of 44 single nucleotide polymorphisms (SNPs), 103 small (≤3 nucleotides) indels, and 1 large (1204 bp) insertion in the Chicago genome with respect to the Nichols genome. To confirm the above findings, Sanger sequencing was performed on most loci carrying differences using DNA from Chicago and the Nichols strain used in the original T. pallidum genome project. A majority of the previously identified differences were found to be due to errors in the published Nichols genome, while the accuracy of the Chicago genome was confirmed. However, 20 SNPs were confirmed between the two genomes, and 16 (80.0%) were found in coding regions, with all being of non-synonymous nature, strongly indicating action of positive selection. Sequencing of 16 genomic loci harboring SNPs in 12 additional T. pallidum strains, (SS14, Bal 3, Bal 7, Bal 9, Sea 81-3, Sea 81-8, Sea 86-1, Sea 87-1, Mexico A, UW231B, UW236B, and UW249C), was used to identify “Chicago-“ or “Nichols -specific” differences. All but one of the 16 SNPs were “Nichols-specific”, with Chicago having identical sequences at these positions to almost all of the additional strains examined. These mutations could reflect differential adaptation of the Nichols strain to the rabbit host or pathoadaptive mutations acquired during human infection. Our findings indicate that SNPs among T. pallidum strains emerge under positive selection and, therefore, are likely to be functional in nature. During infection, the agent of syphilis, Treponema pallidum subsp. pallidum (T. pallidum), successfully evades the host immune defenses and establishes a persistent infection that can cause blindness, paralysis, or even death in some individuals that progress to the tertiary stage of the disease. The study of the Nichols strain of T. pallidum, isolated over a century ago and continually propagated in rabbits, has been paramount to deepen our knowledge on the biology of the agent of syphilis and the pathogenesis of this complex disease. Nonetheless, when the more recent Chicago isolate of T. pallidum is compared to the Nichols strain, significant differences in gene expression, gene conversion rates, and antibody responses against virulence factor candidates are detected during experimental infection. To investigate whether differences at the genomic level between Nichols and Chicago might explain such phenotypic differences, we sequenced the Chicago strain genome and compared it to the previously sequenced T. pallidum Nichols strain. Our findings indicate that the genomic differences between these T. pallidum strains emerge under positive selection, and are likely to be functional in nature, thereby being involved in shaping the phenotypic diversity between the Chicago and Nichols strains.