Treponema pallidum ssp. pallidum is the causative agent of syphilis, a sexually transmitted infection characterized by multi-stage disease and diverse clinical manifestations. Treponema pallidum undergoes rapid vascular dissemination to penetrate tissue, placental, and blood-brain barriers and gain access to distant tissue and organ sites. The rapidity and extent of T. pallidum dissemination is well documented, but the molecular mechanisms that underlie this process have yet to be fully elucidated. Tp0751 is a T. pallidum adhesin that interacts with vascular factors and mediates adherence to endothelial cells under shear flow. This dissertation explores the molecular interactions and functional outcomes of Tp0751-mediated vascular endothelium adhesion. The findings presented herein demonstrate that recombinant Tp0751 adheres to human macrovascular and microvascular endothelial cells, including cerebral brain endothelial cells. This interaction is confirmed using live T. pallidum, where spirochete- endothelial cells interactions are disrupted with Tp0751-specific antiserum. Further, the 67 kDa laminin receptor (LamR) is identified as an endothelial receptor using affinity chromatography coupled with mass spectrometry to isolate and identify Tp0751-interacting proteins from endothelial cells membrane extracts. Notably, LamR is a brain endothelial cell receptor for other neurotropic invasive pathogens. Evaluation of endothelial intercellular junctions reveals that recombinant Tp0751 and live T. pallidum disrupt junctional architecture. However, transwell solute flux assays reveal that Tp0751 and T. pallidum do not alter endothelial barrier integrity. The transendothelial migration of T. pallidum can be partially abrogated with an endocytosis inhibitor, implying a transcellular route for barrier traversal. However, a subpopulation of T. pallidum localizes to intercellular junctions, indicating paracellular traversal may also be employed. These findings enhance our understanding of the mechanics of T. pallidum attachment to endothelial cells and suggest that T. pallidum may use both paracellular and transcellular mechanisms to traverse the vascular endothelium without altering barrier permeability. A more complete understanding of this process will facilitate vaccine development for syphilis. / Graduate / 2020-06-18

<div>The eradication of yaws a neglected tropical disease caused by Treponema pallidum subsp. pertenue, which affects children living in very deprived hard to reach rural communities is constrained by the lack of rapid, accurate diagnosis. I sought to develop a molecular point-of-care test for the diagnosis of yaws. A Loop-mediated isothermal amplification (LAMP) assay with primers targeting the conserved gene, tp0967, with visual detection by lateral flow test strip was developed and optimized. The limit of detection was evaluated while 63 samples from clinical cases of yaws and 5 samples with PCR-confirmed syphilis were used to determine the sensitivity and specificity of the assay compared to the current molecular testing protocol. Reagents were dried in tubes and tested up to 14 days. The developed LAMP assay was found to be optimal when run at 65oC in a water bath for 30 minutes. The limit of detection was 2.7*104 DNA copies/ml. The sensitivity of the LAMP assay using unextracted and DNA extracted samples were 0.67 and 1.00 respectively. None of the syphilis samples tested positive in any of the assays. We show the development of a fast and sensitive LAMP assay for yaws detected by lateral flow test strip. Using extracted DNA, the assay sensitivity is at par with gold standard detection. The assay can be adapted to minimal sample processing required for in-field detection without DNA extraction.</div><div><br></div>

mormaeche@dge.gob.pe / Objective: To assess the seroprevalence of hepatitis B virus (HBV), syphilis, and HIV and associated risk factors in pregnant women and their male partners from six indigenous populations of the Peruvian Amazon Basin. Methods: A cross-sectional study was performed in six indigenous populations from the Peruvian Amazon Basin. Blood samples were obtained and tested for HBV (antibodies to the hepatitis B core antigen (anti-HBc) and hepatitis B surface antigen (HBsAg)), for syphilis (rapid plasma reagin and microhemagglutination assay for Treponema pallidum antibodies), and for HIV (ELISA and indirect immunofluorescence test). A survey was also performed to identify associated risk factors. Results: One thousand two hundred and fifty-one pregnant women and 778 male partners were enrolled in the study. The seroprevalence of anti-HBc in pregnant women was 42.06% (95% confidence interval (CI) 39.28–44.85%) and in their male partners was 54.09% (95% CI 50.32–57.86%). The seroprevalence of HBsAg in pregnant women was 2.11% (95% CI 0.78–3.44%) and in their male partners was 3.98% (95% CI 1.87–6.08%). The seroprevalence of syphilis in pregnant women was 1.60% (95% CI 0.86–2.33%) and in their male partners was 2.44% (95% CI 1.22–3.66%). HIV seroprevalence in pregnant women was 0.16% (95% CI 0.02–0.58%) and in their male partners was 0.29% (95% CI 0.04–1.03%). Sexual risk factors were strongly related to blood markers of syphilis and HBV. Conclusions: Hepatitis B was found to be hyperendemic and strongly related to sexual factors, suggesting an important sexual component in the transmission of the disease in the populations studied. Syphilis was found to have an endemicity in pregnant women above the national level and this may be indicative of high mother-to-child transmission. HIV has started to show its presence in indigenous populations of the Amazon Basin and the results suggest the epidemic is concentrated. / Revisión por pares

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 89-111).

Syphilis is a global public health concern with 36.4 million cases worldwide and 11 million new infections per year. It is a chronic multistage disease caused by the spirochete bacterium Treponema pallidum and is transmitted by sexual contact, direct contact with lesions or vertically from an infected mother to her fetus. T. pallidum is a highly invasive pathogen that rapidly penetrates tight junctions of endothelial cells and disseminates rapidly via the bloodstream to establish widespread infection. Previous investigations conducted in our laboratory identified the surface-exposed adhesin, pallilysin, as a metalloprotease that degrades the host components laminin (major component of the basement membrane lining blood vessels) and fibrinogen (primary component of the coagulation cascade), as well as fibrin clots (function to entrap bacteria and prevent disseminated infection). Furthermore, pallilysin expressed on the surface of the non-invasive spirochete Treponema phagedenis conferred upon this bacterium the ability to degrade fibrin clots. It was hypothesized that pallilysin is integral to the process of T. pallidum dissemination, and interference with its functioning will prevent spread throughout the host and establishment of chronic infection. To test this hypothesis, a two-pronged approach was undertaken during my thesis research. Bioinformatics analyses were used to trace the evolutionary history of pallilysin in an attempt to gain further insight into its role in the pathogenesis of T. pallidum. The sequence conservation of pallilysin was analyzed in the context of its homologues. The bioinformatics analyses revealed homologues in three spirochete genera, namely Treponema, Spirochaeta, and Borrelia, presented in decreasing order of the degree of sequence conservation. The HEXXH motif, part of the active site of the pallilysin metalloprotease, was fully conserved only in T. pallidum and T. paraluiscuniculi, both of which are systemic pathogens. However, the flanking sequences showed a high degree of conservation, especially in the Treponema and Spirochaeta genera. The minimum laminin-binding region of pallilysin identified previously was partially conserved among the treponema and spirochaeta homologues with the highest degree of conservation observed with the homologues from T. paraluiscuniculi and T. phagedenis, as well as among the homologues from the human oral pathogens. In vitro dissemination studies were performed to investigate the dissemination capacity of T. phagedenis heterologously expressing pallilysin. Human Umbilical Vein Endothelial Cells were seeded and grown to confluence on permeable inserts coated with growth factor-reduced Matrigel to create an artificial endothelial barrier. Wild type T. phagedenis, and T. phagedenis transformed either with the pallilysin open reading frame or its empty shuttle vector, were incubated with the barriers under anaerobic conditions. Dissemination across the barrier was assessed as percent traversal by both dark-field microscopic counts of treponemes and real-time quantitative PCR of genomic DNA extracted from the treponemes. The results were inconclusive. However, a traversal trend suggested heterologous expression of pallilysin may facilitate traversal of T. phagedenis across the artificial endothelial barrier. This study presented the first step towards elucidating the role of pallilysin in endothelial monolayer traversal and provided supporting evidence for the role of pallilysin in the widespread dissemination of T. pallidum in vivo. / Graduate

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [63]-80).

Recent bioinformatic analyses identified an operon encoding a potential Type I Secretion System (T1SS) in Treponema pallidum that we hypothesize functions to export key treponemal virulence factors that may contribute to the unique invasiveness and pathogenesis of this spirochete. The membrane fusion protein component (MFP) of T1SSs in other organisms has been shown to play a role in substrate recognition. Hence, the objective of this project is to use the putative MFP, Tp0965, of the potential T. pallidum T1SS to investigate protein-protein interactions with the T. pallidum virulence factor pallilysin (Tp0751) and assess the possibility of the latter being a T1SS substrate. Moreover, protein-protein interactions between Tp0965 and a Treponema phagedenis lysate are investigated with the goal of identifying putative T1SS substrates in this spirochete that could result in the discovery of novel T. pallidum virulence factors via amino acid sequence similarity. Plate-based binding studies and pull-down assays showed a low level of interaction between recombinant Tp0965 and the previously characterized host-component-binding protease, pallilysin, suggesting that the export of this virulence factor could occur via the putative T1SS. Additionally, bioinformatic analyses of the related but cultivable model spirochete T. phagedenis predicted the presence of a potential T1SS homologous to the putative T1SS in T. pallidum. Thus, a more global and unbiased pull-down assay using “bait” Tp0965 and a “prey” T. phagedenis lysate was carried out, followed by mass spectrometric analysis to identify putative novel T1SS substrates with potential homologs in T. pallidum. We successfully identified a T. phagedenis protein, TphBIg, that showed evidence of an interaction with Tp0965. TphBIg seems to possess characteristics of a T1SS substrate suggesting it may be secreted via this system in T. phagedenis. Upon bioinformatic analysis, it was found that TphBIg showed weak amino acid sequence similarity as well as some structural similarity to the T. pallidum protein, Tp0854. Tp0854 is predicted to contain a sialidase and a phosphatase domain with an RTX motif, which is characteristic of some T1SS substrates. Thus, it was hypothesized that if Tp0854 had characteristics of a T1SS, it may interact with Tp0965. Therefore, the phosphatase domain containing the RTX motif was produced recombinantly and plate-based binding studies indeed suggested an interaction with Tp0965, confirming the in silico-predicted interaction. Future experiments to characterize the potential T1SS and substrates in T. pallidum could comprise the functional and structural characterization of the novel putative T1SS substrate, Tp0854. This would include assays to investigate the putative sialidase and phosphatase activities of Tp0854, as well as the identification of Tp0854-Tp0965 interacting sites. Moreover, as a more definite test for T1SS substrate secretion, T. pallidum pallilysin and/or Tp0854 could be expressed heterologously in an E. coli strain harbouring an endogenous T1SS and test for secretion. Similarly, the reconstitution of the T. pallidum putative T1SS in liposomes could be used to further investigate the secretion of pallilysin and/or Tp0854 via this system. Additionally, the optimized unbiased pull-down technique could be further applied to detect more protein-protein interactions within T. pallidum and potentially lead to the identification of more virulence factors that may be secreted via the T1SS. These studies constitute the first investigation of a putative T1SS and substrates within T. pallidum. Thus, insight gained will lead to a better understanding of the mechanisms facilitating T. pallidum host invasion and may reveal new potential vaccine targets to prevent bacterial dissemination and chronic infection. / Graduate

Syfilis är en sexuellt överförbar infektion som orsakas av spiroketen Treponema pallidum subspecies pallidum. Laboratoriediagnostik utgörs i första hand av serologiska test tillsammans med kliniska fynd men på grund av olika faktorer är dessa inte helt pålitliga. Ett flertal olika PCR-metoder utvecklats som påvisar patogenen med hjälp av T-pallidum-specifika gener. En väl studerad gen med hög specificitet är polA-genen som anses vara en mycket robust och känslig metod och lämpar sig väl för att påvisa T. pallidum i kliniska material. Syftet med projektet är att utveckla och optimera en kvalitativ realtids-PCR i singelplex format för verifiering av T. pallidum, för diagnostisering av syfilis. I detta projekt har en realtids-PCR riktad mot polA för detektion av T. pallidum utvecklats. Metodens prestanda utvärderades med en kommersiellt framställd DNA-kontroll med avseende på sensitivitet, specificitet detektions-gräns. När samtliga moment verifierats och fastställts testades metoden på kliniskt material från sår. Den T. pallidum-specifika PCR-analysen visade att det inte förekom korsreaktivitet mot andra agens som förväntas finnas i sår från patient med misstänkt syfilis. Metoden uppvisade en känslighet vid spädning 1:100 (cirka 13 kopior/µl) och en precision på 36,3±0,8 Ct. Det kliniska provet visade på förekomst av T. pallidum dock är känsligheten något sämre än existerande referensmetod. Metoden kan användas i rutindiagnostik av T. pallidum dock bör utfallet från extraktion av DNA från sår studeras ytterligare för att eventuellt öka utbytet och detektera lägre koncentrationer i kliniskt material. / Syphilis is a sexually transmitted infection caused by the spirochete Treponema pallidum subsp. pallidum. Laboratory diagnostics consist primarily of serological tests together with clinical findings for different reasons these methods are not reliable. A variety of PCR methods has been developed that target the pathogen using T. pallidum-specific genes. A well-studied gene with high specificity is the polA gene which is considered very robust and sensitive method and well suited for detection of T. pallidum in clinical materials. The aim of the project is to develop and optimize a qualitative real-time PCR in single-plex format for the verification of T. pallidum, for the diagnosis of syphilis. A real-time PCR targeting polA was developed for detection of T. pallidum. An evaluation of the method's performance was done with a commercially produced DNA control with regards to sensitivity, specificity detection limit and precision. When all test where verified and established, the method was tested on clinical material from ulcers. The results of the T. pallidum-specific PCR-assay showed that there was no cross-reactivity to other agents that are expected to be in ulcers from patients with suspected syphilis. The method showed a sensitivity at dilution 1:100 (about 13 copies/μl) and a precision of 36.3 ± 0.8 Ct.The clinical specimen showed presence of T. pallidum, however, the sensitivity was not as good than the existing reference method. The method can be used in routine diagnostics of T. pallidum, however, the outcome of extraction of DNA from ulcers should be further studied in order to increase the yield and detect lower concentrations in clinical material.

This diploma thesis is dealing with methods of identification genetic variability in sequencing data. The resarch is targeted to bacterial strains of Treponema pallidum. The sequencing was performed by Illumina platform. There is a proposition of method to identificate variable spots in resequenced genomes and their analysis and comparation across all processed genomes.

No / Aim: A diagnostic test that could detect Treponema pallidum antigens in urine would facilitate the prompt diagnosis of syphilis. Materials & methods: Urine from 54 individuals with various clinical stages of syphilis and 6 controls were pooled according to disease stage and interrogated with complementary mass spectrometry techniques to uncover potential syphilis biomarkers. Results & conclusion: In total, 26 unique peptides were uncovered corresponding to four unique T. pallidum proteins that have low genetic sequence similarity to other prokaryotes and human proteins. This is the first account of direct T. pallidum protein detection in human clinical samples using mass spectrometry. The implications of these findings for future diagnostic test development is discussed. Data are available via ProteomeXchange with identifier PXD009707.