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 ß-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.

Treponema pallidum genome sequencing from six continents reveals variability in vaccine candidate genes and dominance of Nichols clade strains in Madagascar.

In spite of its immutable susceptibility to penicillin, Treponema pallidum (T. pallidum) subsp. pallidum continues to cause millions of cases of syphilis each year worldwide, resulting in significant morbidity and mortality and underscoring the urgency of developing an effective vaccine to curtail the spread of the infection. Several technical challenges, including absence of an in vitro culture system until very recently, have hampered efforts to catalog the diversity of strains collected worldwide. Here, we provide near-complete genomes from 196 T. pallidum strains-including 191 T. pallidum subsp. pallidum-sequenced directly from patient samples collected from 8 countries and 6 continents. Maximum likelihood phylogeny revealed that samples from most sites were predominantly SS14 clade. However, 99% (84/85) of the samples from Madagascar formed two of the five distinct Nichols subclades. Although recombination was uncommon in the evolution of modern circulating strains, we found multiple putative recombination events between T. pallidum subsp. pallidum and subsp. endemicum, shaping the genomes of several subclades. Temporal analysis dated the most recent common ancestor of Nichols and SS14 clades to 1717 (95% HPD: 1543-1869), in agreement with other recent studies. Rates of SNP accumulation varied significantly among subclades, particularly among different Nichols subclades, and was associated in the Nichols A subclade with a C394F substitution in TP0380, a ERCC3-like DNA repair helicase. Our data highlight the role played by variation in genes encoding putative surface-exposed outer membrane proteins in defining separate lineages, and provide a critical resource for the design of broadly protective syphilis vaccines targeting surface antigens.

A Novel tp0548 Gene Type of Treponema pallidum Identified in Nanjing, China: Case Report and Review of Literature.

BACKGROUND: The tp0548 gene, hypothesized to encode for an outer-membrane protein, was originally used in the enhanced Centers for Disease Control and Prevention typing for molecular typing of Treponema pallidum. It plays an important role in the molecular epidemiology of Treponema because it is not only an important locus of multiple typing approaches but also suitable for strain typing of multiple Treponema subspecies. METHODS: A 27-year-old Chinese man attended the Institute of Dermatology, Chinese Academy of Medical Sciences Sexually Transmitted Disease Clinic in Nanjing, China, because of a genital ulcer and inguinal lymphadenopathy for 1 week. Workup consisted of microbiological and hematological investigations, and sequences analysis. The aims of this study were to describe a novel tp0548 sequence type "Qn" of this syphilis strain and to review all previously reported novel tp0548 genotypes. RESULTS: We identified a novel tp0548 gene type in a genital ulcer in a patient with primary syphilis in Nanjing, China. Using sequence alignment, we further found that this novel sequence was closely similar to "Q." Following the nomenclature used in the enhanced Centers for Disease Control and Prevention typing methodology, the letters "Qn" was assigned to the new sequence type. CONCLUSION: The novel tp0548 sequence type of T. pallidum not only expands the database up to 27 different sequence types but also indicates the substantial genetic diversity of the tp0548 gene sequence.

MiR-223-3p inhibits rTp17-induced inflammasome activation and pyroptosis by targeting NLRP3.

The incidence of syphilis caused by Treponema pallidum subsp pallidum (T pallidum) infection is accompanied by inflammatory injuries of vascular endothelial cells. Studies have revealed that T pallidum infection could induce inflammasome activation and pyroptosis in macrophages. MicroRNA-223-3p (miR-223-3p) was reported to be a negative regulator in inflammatory diseases. The present study aimed to explore whether miR-223-3p regulates T pallidum-induced inflammasome activation and pyroptosis in vascular endothelial cells, and determine the mechanisms which underlie this process. MiR-223-3p levels in syphilis and control samples were determined. The biological function of miR-223-3p in the NLRP3 inflammasome and pyroptosis was evaluated in T pallidum-infected human umbilical vein endothelial cells (HUVECs). We observed a dramatic decrease in miR-223-3p levels in syphilis patients (n = 20) when compared to healthy controls (n = 20). Moreover, miR-223-3p showed a notable inhibitory effect on recombinant Tp17 (rTP17)-induced caspase-1 activation, resulting in decrease in IL-1ß production and pyroptosis, which was accompanied by the release of lactate dehydrogenase (LDH) in HUVECs. Additionally, the dual-luciferase assay confirmed that NLRP3 is a direct target of miR-223-3p. Moreover, NLRP3 overexpression or knockdown largely blocked the effects of miR-223-3p on T pallidum-induced inflammasome activation and pyroptosis in HUVECs. Most importantly, a notable negative correlation was observed between miR-223-3p and NLRP3, caspase-1, and IL-1ß, respectively, in the serum of syphilis patients and healthy controls. Taken together, our results reveal that miR-223-3p targets NLRP3 to suppress inflammasome activation and pyroptosis in T pallidum-infected endothelial cells, implying that miR-223-3p could be a potential target for syphilis patients.