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

Sequence Variation of Rare Outer Membrane Protein β-Barrel Domains in Clinical Strains Provides Insights into the Evolution of Treponema pallidum subsp. pallidum, the Syphilis Spirochete

In recent years, considerable progress has been made in topologically and functionally characterizing integral outer membrane proteins (OMPs) of Treponema pallidum subspecies pallidum, the syphilis spirochete, and identifying its surface-exposed β-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are known to be highly variable. We examined the sequence diversity of β-barrel-encoding regions of tprC, tprD, and bamA in 31 specimens from Cali, Colombia; San Francisco, California; and the Czech Republic and compared them to allelic variants in the 41 reference genomes in the NCBI database. To establish a phylogenetic framework, we used T. pallidum 0548 (tp0548) genotyping and tp0558 sequences to assign strains to the Nichols or SS14 clades. We found that (i) β-barrels in clinical strains could be grouped according to allelic variants in T. pallidum subsp. pallidum reference genomes; (ii) for all three OMP loci, clinical strains within the Nichols or SS14 clades often harbored β-barrel variants that differed from the Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in predicted extracellular loops containing B-cell epitopes. On the basis of structural models, nonconservative amino acid substitutions in predicted transmembrane β-strands of T. pallidum repeat C (TprC) and TprD2 could give rise to functional differences in their porin channels. OMP profiles of some clinical strains were mosaics of different reference strains and did not correlate with results from enhanced molecular typing. Our observations suggest that human host selection pressures drive T. pallidum subsp. pallidum OMP diversity and that genetic exchange contributes to the evolutionary biology of T. pallidum subsp. pallidum. They also set the stage for topology-based analysis of antibody responses to OMPs and help frame strategies for syphilis vaccine development.Despite recent progress characterizing outer membrane proteins (OMPs) of Treponema pallidum, little is known about how their surface-exposed, β-barrel-forming domains vary among strains circulating within high-risk populations. In this study, sequences for the β-barrel-encoding regions of three OMP loci, tprC, tprD, and bamA, in T. pallidum subsp. pallidum isolates from a large number of patient specimens from geographically disparate sites were examined. Structural models predict that sequence variation within β-barrel domains occurs predominantly within predicted extracellular loops. Amino acid substitutions in predicted transmembrane strands that could potentially affect porin channel function were also noted. Our findings suggest that selection pressures exerted within human populations drive T. pallidum subsp. pallidum OMP diversity and that recombination at OMP loci contributes to the evolutionary biology of syphilis spirochetes. These results also set the stage for topology-based analysis of antibody responses that promote clearance of T. pallidum subsp. pallidum and frame strategies for vaccine development based upon conserved OMP extracellular loops

Immune Evasion and Recognition of the Syphilis Spirochete in Blood and Skin of Secondary Syphilis Patients: Two Immunologically Distinct Compartments

<div><h3>Background</h3><p>The clinical syndrome associated with secondary syphilis (SS) reflects the propensity of <em>Treponema pallidum</em> (<em>Tp</em>) to escape immune recognition while simultaneously inducing inflammation.</p> <h3>Methods</h3><p>To better understand the duality of immune evasion and immune recognition in human syphilis, herein we used a combination of flow cytometry, immunohistochemistry (IHC), and transcriptional profiling to study the immune response in the blood and skin of 27 HIV(-) SS patients in relation to spirochetal burdens. <em>Ex vivo</em> opsonophagocytosis assays using human syphilitic sera (HSS) were performed to model spirochete-monocyte/macrophage interactions <em>in vivo</em>.</p> <h3>Results</h3><p>Despite the presence of low-level spirochetemia, as well as immunophenotypic changes suggestive of monocyte activation, we did not detect systemic cytokine production. SS subjects had substantial decreases in circulating DCs and in IFNγ-producing and cytotoxic NK-cells, along with an emergent CD56−/CD16+ NK-cell subset in blood. Skin lesions, which had visible <em>Tp</em> by IHC and substantial amounts of <em>Tp</em>-DNA, had large numbers of macrophages (CD68+), a relative increase in CD8+ T-cells over CD4+ T-cells and were enriched for CD56+ NK-cells. Skin lesions contained transcripts for cytokines (IFN-γ, TNF-α), chemokines (CCL2, CXCL10), macrophage and DC activation markers (CD40, CD86), Fc-mediated phagocytosis receptors (FcγRI, FcγR3), IFN-β and effector molecules associated with CD8 and NK-cell cytotoxic responses. While HSS promoted uptake of <em>Tp</em> in conjunction with monocyte activation, most spirochetes were not internalized.</p> <h3>Conclusions</h3><p>Our findings support the importance of macrophage driven opsonophagocytosis and cell mediated immunity in treponemal clearance, while suggesting that the balance between phagocytic uptake and evasion is influenced by the relative burdens of bacteria in blood and skin and the presence of <em>Tp</em> subpopulations with differential capacities for binding opsonic antibodies. They also bring to light the extent of the systemic innate and adaptive immunologic abnormalities that define the secondary stage of the disease, which in the skin of patients trends towards a T-cell cytolytic response.</p> </div

Emergence of a CD56-negative NK-cell population in secondary syphilis (SS) patients.

<p>(<b>A</b>) The CD56^negative CD16+ NK-cells subset in untreated SS patients are shown by the black arrows. (<b>B</b>) Significant increases (* <i>p</i> = 0.003) in CD56^negative CD16+ NK-cell population was seen in SS patients. This anomaly was not present in healthy controls.</p

<i>T. pallidum</i> (<i>Tp</i>) uptake and recovery by IFA.

<p>Purified human monocytes obtained from healthy controls were stimulated with fresh <i>Tp</i> Nichols strain (MOI 1∶1, 10∶1 and 30∶1) were incubated for 8-hours alone or where indicated in the presence of 10% heat inactivated normal human serum (NHS) or human syphilitic serum (HSS). (<b>A</b>) Percentage of phagocytosed <i>Tp</i> was greater when HSS was present. (<b>B</b>) Percentage of non-phagocytosed <i>Tp</i> which were recovered in supernatants at the end of incubation time is shown in each of the two graphs. Spirochetal recovery was substantially higher in the absence of HSS; nonetheless more than half of the bacteria avoided recognition and uptake despite the presence of HSS (<i>p</i> values shown correspond to statistical comparisons between groups by ANOVA). (<b>C</b>) Percentage of spirochetes recovered was similar in the presence of HSS at three different MOIs.</p

Clinical and laboratory characteristics of secondary syphilis patients.

*<p>Diagnostic PCR results previously published in PLoS NTD (Ref 8).</p

Spirochetal clusters are present in secondary syphilis skin lesions.

<p>Representative skin biopsy from a posterior neck secondary syphilis (SS) lesion was processed for IHC. (<b>A/B</b>) H&E stain of SS lesions. (<b>C/D</b>) IHC staining reveals abundant spirochetes embedded within a mixed cellular inflammatory infiltrate (shown in the red box) in the papillary dermis. The blue arrow points to a tissue histiocyte and the read arrows to two dermal lymphocytes.</p

Cell surface activation markers in secondary syphilis patient monocytes.

<p>Cell surface activation markers were examined in monocytes from secondary syphilis (SS) patients before (Pre-Tx) and around 60 days after penicillin treatment (Post-Tx) and compared to healthy controls. (<b>A</b>) A modest but significant decrease in CD40 MFI was evident between paired acute and convalescent samples obtained from SS patients before and after treatment. (<b>B</b>) Significant increases in CD14 MFI expression were observed between syphilis patients and healthy volunteers prior to antibiotic treatment and between pre- and post-penicillin treatment (<i>p</i> values are shown in the figure).</p