High-throughput nanopore sequencing of Treponema pallidum tandem repeat genes arp and tp0470 reveals clade-specific patterns and recapitulates global whole genome phylogeny

Sequencing of most Treponema pallidum genomes excludes repeat regions in tp0470 and the tp0433 gene, encoding the acidic repeat protein (arp). As a first step to understanding the evolution and function of these genes and the proteins they encode, we developed a protocol to nanopore sequence tp0470 and arp genes from 212 clinical samples collected from ten countries on six continents. Both tp0470 and arp repeat structures recapitulate the whole genome phylogeny, with subclade-specific patterns emerging. The number of tp0470 repeats is on average appears to be higher in Nichols-like clade strains than in SS14-like clade strains. Consistent with previous studies, we found that 14-repeat arp sequences predominate across both major clades, but the combination and order of repeat type varies among subclades, with many arp sequence variants limited to a single subclade. Although strains that were closely related by whole genome sequencing frequently had the same arp repeat length, this was not always the case. Structural modeling of TP0470 suggested that the eight residue repeats form an extended α-helix, predicted to be periplasmic. Modeling of the ARP revealed a C-terminal sporulation-related repeat (SPOR) domain, predicted to bind denuded peptidoglycan, with repeat regions possibly incorporated into a highly charged β-sheet. Outside of the repeats, all TP0470 and ARP amino acid sequences were identical. Together, our data, along with functional considerations, suggests that both TP0470 and ARP proteins may be involved in T. pallidum cell envelope remodeling and homeostasis, with their highly plastic repeat regions playing as-yet-undetermined roles

Efficacy of linezolid on Treponema pallidum, the syphilis agent : A preclinical study

Penicillin G, the current standard treatment for syphilis, has important drawbacks, but virtually no preclinical or clinical studies have been performed to identify viable alternatives. We tested, both in vitro and in vivo, three marketed antibiotics with adequate pharmacological properties to treat syphilis. We used an in vitro culturing system of T. pallidum to perform drug susceptibility testing and applied quantitative PCR targeting the tp0574 gene to measure bacterial growth. To confirm in vivo efficacy, fifteen rabbits were infected intradermally with T. pallidum at eight sites each and randomly allocated to an experimental treatment (linezolid, moxifloxacin, clofazimine) or a control arm (benzathine penicillin G [BPG], untreated). The primary outcome was treatment efficacy defined as the time to lesion healing measured from the date of treatment start. Secondary outcomes were absence of treponemes or treponemal mRNA in injection sites, absence of seroconversion, and cerebrospinal fluid (CSF) abnormalities and negative rabbit infectivity tests (RIT). Linezolid showed in vitro bactericidal activity at concentrations of 0.5 µg/mL or higher. When administered orally to experimentally infected rabbits, it induced healing of early lesions at a time similar to BPG (hazard ratio 3.84; 95% CI 2.05-7.17; p < 0.0001 compared to untreated controls). In linezolid-treated animals, dark-field microscopy and qPCR assessment showed no presence of treponemes after day 3 post-treatment start, serologic test did not convert to positive, CSF had no abnormalities, and RIT was negative. Moxifloxacin and clofazimine failed to inhibit bacterial growth in vitro and could not cure the infection in the rabbit model. Linezolid, a low-cost oxazolidinone, has in vitro and in vivo activity against T. pallidum, with efficacy similar to BPG in treating treponemal lesions in the animal model. Our findings warrant further research to assess the efficacy of linezolid as an alternative to penicillin G to treat syphilis in human clinical trials

Treponema pallidum subsp. pallidum with an Artificially impaired TprK antigenic variation system is attenuated in the Rabbit model of syphilis.

BackgroundThe TprK protein of the syphilis agent, Treponema pallidum subsp. pallidum (T. pallidum), undergoes antigenic variation in seven discrete variable (V) regions via non-reciprocal segmental gene conversion. These recombination events transfer information from a repertoire of 53 silent chromosomal donor cassettes (DCs) into the single tprK expression site to continually generate TprK variants. Several lines of research developed over the last two decades support the theory that this mechanism is central to T. pallidum's ability for immune avoidance and persistence in the host. Structural and modeling data, for example, identify TprK as an integral outer membrane porin with the V regions exposed on the pathogen's surface. Furthermore, infection-induced antibodies preferentially target the V regions rather than the predicted β-barrel scaffolding, and sequence variation abrogates the binding of antibodies elicited by antigenically different V regions. Here, we engineered a T. pallidum strain to impair its ability to vary TprK and assessed its virulence in the rabbit model of syphilis.Principal findingsA suicide vector was transformed into the wild-type (WT) SS14 T. pallidum isolate to eliminate 96% of its tprK DCs. The resulting SS14-DCKO strain exhibited an in vitro growth rate identical to the untransformed strain, supporting that the elimination of the DCs did not affect strain viability in absence of immune pressure. In rabbits injected intradermally with the SS14-DCKO strain, generation of new TprK sequences was impaired, and the animals developed attenuated lesions with a significantly reduced treponemal burden compared to control animals. During infection, clearance of V region variants originally in the inoculum mirrored the generation of antibodies to these variants, although no new variants were generated in the SS14-DCKO strain to overcome immune pressure. Naïve rabbits that received lymph node extracts from animals infected with the SS14-DCKO strain remained uninfected.ConclusionThese data further support the critical role of TprK in T. pallidum virulence and persistence during infection

Transcriptional and immunological analysis of the putative outer membrane protein and vaccine candidate TprL of Treponema pallidum.

BackgroundAn effective syphilis vaccine should elicit antibodies to Treponema pallidum subsp. pallidum (T. p. pallidum) surface antigens to induce pathogen clearance through opsonophagocytosis. Although the combination of bioinformatics, structural, and functional analyses of T. p. pallidum genes to identify putative outer membrane proteins (OMPs) resulted in a list of potential vaccine candidates, still very little is known about whether and how transcription of these genes is regulated during infection. This knowledge gap is a limitation to vaccine design, as immunity generated to an antigen that can be down-regulated or even silenced at the transcriptional level without affecting virulence would not induce clearance of the pathogen, hence allowing disease progression.Principal findingsWe report here that tp1031, the T. p. pallidum gene encoding the putative OMP and vaccine candidate TprL is differentially expressed in several T. p. pallidum strains, suggesting transcriptional regulation. Experimental identification of the tprL transcriptional start site revealed that a homopolymeric G sequence of varying length resides within the tprL promoter and that its length affects promoter activity compatible with phase variation. Conversely, in the closely related pathogen T. p. subsp. pertenue, the agent of yaws, where a naturally-occurring deletion has eliminated the tprL promoter region, elements necessary for protein synthesis, and part of the gene ORF, tprL transcription level are negligible compared to T. p. pallidum strains. Accordingly, the humoral response to TprL is absent in yaws-infected laboratory animals and patients compared to syphilis-infected subjects.ConclusionThe ability of T. p. pallidum to stochastically vary tprL expression should be considered in any vaccine development effort that includes this antigen. The role of phase variation in contributing to T. p. pallidum antigenic diversity should be further studied

Barcoded primers used in this study for <i>tprk</i> deep sequencing.

Barcoded primers used in this study for tprk deep sequencing.</p

Confirmation of <i>kan</i>^R cassette integration using ddPCR and WGS, and message quantifications.

(A) Ratios between the kanR, dnaA (tp0001), and tp0127 (contained within the deleted DC) copy number determined by droplet digital PCR (ddPCR) on template DNA from the SS14-DCKO and parent SS14 strain harvested at passage #2–5 post-transformation. The kanR:dnaA ratio for the parent SS14 was zero at each passage. (B) Message quantification of tprK (normalized to tp0574) in SS14-DCKO and parent SS14 strain propagated in vitro showing comparable expression of tprK between the control and transformed strain. Significance (pt-test. (C-G) Whole-genome sequencing of the SS14-DCKO and WT strains (passage #9) post-transformation. (C) SS14-DCKO reads assembled to the WT genome reference (NC_021508.1/CP004011.1) showing a gap where the DC locus previously was. (D) SS14-DCKO reads assembled to the SS14-DCKO reference genome, where the DC locus was replaced in silico with the kanR cassette sequence showing reads aligning to the cassette sequence. (E) Schematic of the 51 deleted DCs, color-coded based on their target V region and the kanR cassette (tp0574 promoter- kanR) that replaced the DCs. (F) Reads from the WT SS14 genome aligned to the SS14 reference genome showing the integrity of the DC locus. (G) Reads from the WT genome aligned to the SS14-DCKO reference genome showing a gap where the kanR cassette is located. (H-I) Messsage quantification of the tp0131 and kanR genes in the WT and the SS14-DCKO strain showing lack of transcriptional polar effects due to the substitution of the DCs. mRNA was reverse-transcribed using either gene-specific primers (GSP, white bars) or random hexamers (RE; black bars); ns: not significant.</p

TprK (sequence AIP85979.1) is predicted by AlphaFold2 to fold into a 20-strands beta-barrel structures with 10 surface-exposed loops.

Variable loops (V1-V7) are highlighted in colour. Conserved loops (C1-C3) are in white, and β-scaffolding are in light blue. OM: outer membrane; PS: periplasmic space.</p

Heatmap of sequence diversity in TprK V4.

Deep sequencing of TprK V4 region showing persistence of inoculum variants and generation of non-inoculum variants in samples collected overtime from rabbits infected with the WT SS14 strain (“WT”-labeled samples, left side of the map) and form rabbits infected with the SS14-DCKO strain (“KO”-labeled samples, right side of the map), separated by a bold vertical dashed line. In each map, inoculum sequences for the WT strain are labeled as “WT Inoc B00 Day 00”, where WT Inoc indicates WT inoculum treponemes, B00 indicates that the sample was not obtained from a biopsy, and Day 00 indicates the experiment’s time 0. The same nomenclature was adopted for the SS14-DCKO inoculum, with the exception that KO replaced WT. Samples collected post-inoculation report rabbit number (R#), biopsy number (B#), and day post-inoculation the sample was obtained (Day#). Light gray dashed lines separate individual animals. Missing samples did not yield data. The same heatmap in interactive format is available at https://github.com/greninger-lab/Impaired-TprK-Antigenic-Variation. Peptide sequences and prevalence are also reported in S2 Table. (PDF)</p

Primers used in this study.

BackgroundThe TprK protein of the syphilis agent, Treponema pallidum subsp. pallidum (T. pallidum), undergoes antigenic variation in seven discrete variable (V) regions via non-reciprocal segmental gene conversion. These recombination events transfer information from a repertoire of 53 silent chromosomal donor cassettes (DCs) into the single tprK expression site to continually generate TprK variants. Several lines of research developed over the last two decades support the theory that this mechanism is central to T. pallidum’s ability for immune avoidance and persistence in the host. Structural and modeling data, for example, identify TprK as an integral outer membrane porin with the V regions exposed on the pathogen’s surface. Furthermore, infection-induced antibodies preferentially target the V regions rather than the predicted β-barrel scaffolding, and sequence variation abrogates the binding of antibodies elicited by antigenically different V regions. Here, we engineered a T. pallidum strain to impair its ability to vary TprK and assessed its virulence in the rabbit model of syphilis.Principal findingsA suicide vector was transformed into the wild-type (WT) SS14 T. pallidum isolate to eliminate 96% of its tprK DCs. The resulting SS14-DCKO strain exhibited an in vitro growth rate identical to the untransformed strain, supporting that the elimination of the DCs did not affect strain viability in absence of immune pressure. In rabbits injected intradermally with the SS14-DCKO strain, generation of new TprK sequences was impaired, and the animals developed attenuated lesions with a significantly reduced treponemal burden compared to control animals. During infection, clearance of V region variants originally in the inoculum mirrored the generation of antibodies to these variants, although no new variants were generated in the SS14-DCKO strain to overcome immune pressure. Naïve rabbits that received lymph node extracts from animals infected with the SS14-DCKO strain remained uninfected.ConclusionThese data further support the critical role of TprK in T. pallidum virulence and persistence during infection.</div

Heatmap of sequence diversity in TprK V3.

Deep sequencing of TprK V3 region showing persistence of inoculum variants and generation of non-inoculum variants in samples collected overtime from rabbits infected with the WT SS14 strain (“WT”-labeled samples, left side of the map) and form rabbits infected with the SS14-DCKO strain (“KO”-labeled samples, right side of the map), separated by a bold vertical dashed line. In each map, inoculum sequences for the WT strain are labeled as “WT Inoc B00 Day 00”, where WT Inoc indicates WT inoculum treponemes, B00 indicates that the sample was not obtained from a biopsy, and Day 00 indicates the experiment’s time 0. The same nomenclature was adopted for the SS14-DCKO inoculum, with the exception that KO replaced WT. Samples collected post-inoculation report rabbit number (R#), biopsy number (B#), and day post-inoculation the sample was obtained (Day#). Light gray dashed lines separate individual animals. Missing samples did not yield data. The same heatmap in interactive format is available at https://github.com/greninger-lab/Impaired-TprK-Antigenic-Variation. Peptide sequences and prevalence are also reported in S2 Table. (PDF)</p