In-Depth Proteome Coverage of In Vitro-Cultured Treponema pallidum and Quantitative Comparison Analyses with In Vivo-Grown Treponemes.

Previous mass spectrometry (MS)-based global proteomics studies have detected a combined total of 86% of all Treponema pallidum proteins under infection conditions (in vivo-grown T. pallidum). Recently, a method was developed for the long-term culture of T. pallidum under in vitro conditions (in vitro-cultured T. pallidum). Herein, we used our previously reported optimized MS-based proteomics approach to characterize the T. pallidum global protein expression profile under in vitro culture conditions. These analyses provided a proteome coverage of 94%, which extends the combined T. pallidum proteome coverage from the previously reported 86% to a new combined total of 95%. This study provides a more complete understanding of the protein repertoire of T. pallidum. Further, comparison of the in vitro-expressed proteome with the previously determined in vivo-expressed proteome identifies only a few proteomic changes between the two growth conditions, reinforcing the suitability of in vitro-cultured T. pallidum as an alternative to rabbit-based treponemal growth. The MS proteomics data have been deposited in the MassIVE repository with the data set identifier MSV000093603 (ProteomeXchange identifier PXD047625).

The outer membrane protein Tp92 of Treponema pallidum delays human neutrophil apoptosis via the ERK, PI3K/Akt, and NF-κB pathways.

Syphilis is a persistent sexually transmitted disease caused by infiltration of the elusive pathogen Treponema pallidum. Despite the prevalence of human polymorphonuclear neutrophils (hPMNs) within cutaneous lesions, which are characteristic of incipient syphilis, their role in T. pallidum infection remains unclear. Tp92 is the only T. pallidum helical outer membrane protein that exhibits structural features similar to those of outer membrane proteins in other gram-negative bacteria. However, the functional mechanism of this protein in immune cells remains unclear. Neutrophils are short-lived cells that undergo innate apoptosis in response to external stimuli that typically influence this process. In this study, we determined that Tp92 impedes the activation of procaspase-3 via the ERK MAPK, PI3K/Akt, and NF-κB signaling pathways, consequently suppressing caspase-3 activity within hPMNs, and thereby preventing hPMNs apoptosis. Furthermore, Tp92 could also modulate hPMNs apoptosis by enhancing the expression of the anti-apoptotic protein Mcl-1, stimulating IL-8 secretion, and preserving the mitochondrial membrane potential. These findings provide valuable insights into the molecular mechanisms underlying T. pallidum infection and suggest potential therapeutic targets for syphilis treatment.

Treponema pallidum recombinant protein Tp47 enhanced interleukin-6 secretion in human dermal fibroblasts through the toll-like receptor 2 via the p38, PI3K/Akt, and NF-κB signalling pathways.

Interleukin-6 (IL-6) is a multi-effective cytokine involved in multiple immune responses. Whether fibroblasts also turn out to be a cytokine IL-6 factory during interaction with Treponema pallidum is not yet understood. To explore whether fibroblasts participate in inflammation due to syphilis, a series of experiments were performed to explore the role of T. pallidum lipoprotein Tp47 in IL-6 production in human dermal fibroblasts. The Toll-like receptor 2 (TLR2) and participating signalling pathways in this process were also evaluated. The results showed that the expressions of IL-6 and the protein levels of TLR2 in fibroblasts were upregulated after stimulation with Tp47, and this effect was impeded by the TLR2 inhibitor C29. In addition, Tp47 promoted the phosphorylation of p38, PI3K/Akt, and nuclear factor-kappaB (NF-κB), and the translocation of NF-κB in fibroblasts. Moreover, p38, PI3K, and NF-κB inhibitors significantly reduced IL-6 production in fibroblasts stimulated with Tp47. Furthermore, the TLR2 inhibitor C29 inhibited the phosphorylation of p38, Akt, and NF-κB, and the translocation of NF-κB in fibroblasts. In conclusion, our results showed that Tp47 enhanced IL-6 secretion in human dermal fibroblasts through TLR2 via p38, PI3K/Akt, and NF-κB signalling pathways. These findings contribute to our understanding of syphilis inflammation.

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.

Lights and Shadows of TORCH Infection Proteomics.

Congenital abnormalities cause serious fetal consequences. The term TORCH is used to designate the most common perinatal infections, where: (T) refers to toxoplasmosis, (O) means "others" and includes syphilis, varicella-zoster, parvovirus B19, zika virus (ZIKV), and malaria among others, (R) refers to rubella, (C) relates to cytomegalovirus infection, and (H) to herpes simplex virus infections. Among the main abnormalities identified in neonates exposed to congenital infections are central nervous system (CNS) damage, microcephaly, hearing loss, and ophthalmological impairment, all requiring regular follow-up to monitor its progression. Protein changes such as mutations, post-translational modifications, abundance, structure, and function may indicate a pathological condition before the onset of the first symptoms, allowing early diagnosis and understanding of a particular disease or infection. The term "proteomics" is defined as the science that studies the proteome, which consists of the total protein content of a cell, tissue or organism in a given space and time, including post-translational modifications (PTMs) and interactions between proteins. Currently, quantitative bottom-up proteomic strategies allow rapid and high throughput characterization of complex biological mixtures. Investigating proteome modulation during host-pathogen interaction helps in elucidating the mechanisms of infection and in predicting disease progression. This "molecular battle" between host and pathogen is a key to identify drug targets and diagnostic markers. Here, we conducted a survey on proteomic techniques applied to congenital diseases classified in the terminology "TORCH", including toxoplasmosis, ZIKV, malaria, syphilis, human immunodeficiency virus (HIV), herpes simplex virus (HSV) and human cytomegalovirus (HCVM). We have highlighted proteins and/or protein complexes actively involved in the infection. Most of the proteomic studies reported have been performed in cell line models, and the evaluation of tissues (brain, muscle, and placenta) and biofluids (plasma, serum and urine) in animal models is still underexplored. Moreover, there are a plethora of studies focusing on the pathogen or the host without considering the triad mother-fetus-pathogen as a dynamic and interconnected system.

Exosomal miR-146a-5p from Treponema pallidum-stimulated macrophages reduces endothelial cells permeability and monocyte transendothelial migration by targeting JAM-C.

Exosomal microRNAs (miRNAs) transferred between cells have been implicated in modulating the host immune response in microbial infections. In this study, we isolated exosomes from Treponema pallidum (T. pallidum)-stimulated macrophages and detected differential exosomal miRNA expression using both microarrays, and RT-qPCR. A total of 65 differentially expressed miRNAs (35 upregulated and 30 downregulated) were identified. Of all identified miRNAs, miR-146a-5p was one of the most significantly changed miRNAs with high expression in exosomes from T. pallidum-stimulated macrophages. Furthermore, we isolated plasma exosomes from early syphilis patients and healthy controls, and confirmed miR-146a-5p upregulation in the former group. We also show that exosomal miR-146a-5p is efficiently transported into endothelial cells, reducing monocyte transendothelial migration and endothelial permeability by targeting junctional adhesion molecule C (JAM-C). Luciferase reporter assays confirmed binding of exosomal miR-146a-5p to the 3'untranslated region (3'UTR) of JAM-C. We then demonstrated that also exosomes derived from macrophages stimulated by T. pallidum expressed high levels of miR-146a-5p which could be delivered to endothelial cells, and decreased monocyte transendothelial migration by targeting JAM-C. Overall, this work provides novel insights into the mechanism by which T. pallidum hampers inflammatory reactions of the host via a blockade of leukocytes transendothelial migration and endothelial permeability.

The N-terminal D1 domain of Treponema pallidum flagellin binding to TLR5 is required but not sufficient in activation of TLR5.

Syphilis is a chronic bacterial infection caused by Treponema pallidum (T pallidum) and the pathogenesis that T pallidum infection induces immunopathological damages in skin and other tissues remains unclear. We have previously reported that recombinant flagellins of T pallidum can elicit IL-6 and IL-8 transcriptions via TLR5 pathway. To identify the domains which induced the pro-inflammatory activity and the importance of the interactions between TLR5 and domains, homology-based modelling and comparative structural analyses revealed that Tpflagellins can combine with TLR5 directly. Deletion mutations showed that the ND1 domain binding to TLR5 is required but not sufficient in TLR5 activation. Moreover, site-directed mutagenesis analysis indicated that the arginine residue (Tpflagellins R89) of the ND1 domain and its adjacent residues (Tpflagellins L93 and E113) constitute a hot spot that elicits IL-6, IL-8 transcriptions and TLR5 activation, and affects the binding of Tpflagellins to TLR5. Taken together, these results give insight into the pathogenesis of T pallidum and may contribute to the future design of Tpflagellins-based therapeutics and syphilis vaccine.

Analysis of host cell binding specificity mediated by the Tp0136 adhesin of the syphilis agent Treponema pallidum subsp. pallidum.

BACKGROUND: Syphilis affects approximately 11 million people each year globally, and is the third most prevalent sexually transmitted bacterial infection in the United States. Inability to independently culture and genetically manipulate Treponema pallidum subsp. pallidum, the causative agent of this disease, has hindered our understanding of the molecular mechanisms of syphilis pathogenesis. Here, we used the non-infectious and poorly adherent B314 strain of the Lyme disease-causing spirochete, Borrelia burgdorferi, to express two variants of a known fibronectin-binding adhesin, Tp0136, from T. pallidum SS14 and Nichols strains. Using this surrogate system, we investigated the ability of Tp0136 in facilitating differential binding to mammalian cell lines offering insight into the possible role of this virulence factor in colonization of specific tissues by T. pallidum during infection. PRINCIPAL FINDINGS: Expression of Tp0136 could be detected on the surface of B. burgdorferi by indirect immunofluorescence assay using sera from a secondary syphilis patient that does not react with intact B314 spirochetes transformed with the empty vector. Increase in Tp0136-mediated adherence of B314 strain to human epithelial HEK293 cells was observed with comparable levels of binding exhibited by both Tp0136 alleles. Adherence of Tp0136-expressing B314 was highest to epithelial HEK293 and C6 glioma cells. Gain in binding of B314 strain expressing Tp0136 to purified fibronectin and poor binding of these spirochetes to the fibronectin-deficient cell line (HEp-2) indicated that Tp0136 interaction with this host receptor plays an important role in spirochetal attachment to mammalian cells. Furthermore, preincubation of these cell lines with fibronectin-binding peptide from Staphylococcus aureus FnbA-2 protein significantly inhibited binding of B314 expressing Tp0136. CONCLUSIONS: Our results show that Tp0136 facilitates differential level of binding to cell lines representing various host tissues, which highlights the importance of this protein in colonization of human organs by T. pallidum and resulting syphilis pathogenesis.

Recombinant Treponema pallidum protein Tp47 promotes the migration and adherence of THP-1 cells to human dermal vascular smooth muscle cells by inducing MCP-1 and ICAM-1 expression.

Vascular inflammation is a complex and multifactorial pathophysiological process that plays a crucial role in all stages of syphilis and is responsible for tissue damage. Little is known about the interactions of infiltrating immunocytes with human dermal vascular smooth muscle cells (HDVSMCs) in arterioles during the immunopathogenesis of syphilis. The Treponema pallidum subsp. pallidum membrane protein Tp47 is considered a major inducer of inflammation initiation and development. In this study, we demonstrated that Tp47 promoted the migration and adhesion of THP-1 cells to HDVSMCs. Furthermore, Tp47 increased monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1) mRNA and protein expression levels in a dose- and time-dependent manner. The migration and adhesion of THP-1 cells to HDVSMCs were significantly suppressed by anti-MCP-1 and anti-ICAM-1 neutralizing antibodies, respectively. Further studies revealed that treatment of HDVSMCs with Tp47 activated the PI3K/Akt, p38 MAPK and NF-κB signalling pathways. Inhibition of PI3K/Akt, p38 MAPK and NF-κB suppressed the MCP-1 and ICAM-1 expression induced by Tp47. In addition, the migration and adhesion of THP-1 cells to Tp47-treated HDVSMCs were significantly decreased by pretreatment with PI3K/Akt, p38 MAPK and NF-κB inhibitors. These findings demonstrate that Tp47 promotes the migration and adherence of THP-1 cells to HDVSMCs by inducing MCP-1 and ICAM-1 expression, which is mediated by activation of the PI3K/Akt, p38 MAPK and NF-κB pathways. This study provides a novel potential therapeutic strategy for controlling the vascular inflammatory response in syphilis patients.

Integrated point-of-care testing (POCT) of HIV, syphilis, malaria and anaemia in antenatal clinics in western Kenya: A longitudinal implementation study.

BACKGROUND: In sub-Saharan Africa, HIV, syphilis, malaria and anaemia are leading preventable causes of adverse pregnancy outcomes. In Kenya, policy states women should be tested for all four conditions (malaria only if febrile) at first antenatal care (ANC) visit. In practice, while HIV screening is conducted, coverage of screening for the others is suboptimal and early pregnancy management of illnesses is compromised. This is particularly evident at rural dispensaries that lack laboratories and have parallel programmes for HIV, reproductive health and malaria, resulting in fractured and inadequate care for women. METHODS: A longitudinal eight-month implementation study integrating point-of-care diagnostic tests for the four conditions into routine ANC was conducted in seven purposively selected dispensaries in western Kenya. Testing proficiency of healthcare workers was observed at initial training and at three monthly intervals thereafter. Adoption of testing was compared using ANC register data 8.5 months before and eight months during the intervention. Fidelity to clinical management guidelines was determined by client exit interviews with success defined as ≥90% adherence. FINDINGS: For first ANC visits at baseline (n = 529), testing rates were unavailable for malaria, low for syphilis (4.3%) and anaemia (27.8%), and near universal for HIV (99%). During intervention, over 95% of first attendees (n = 586) completed four tests and of those tested positive, 70.6% received penicillin or erythromycin for syphilis, 65.5% and 48.3% received cotrimoxazole and antiretrovirals respectively for HIV, and 76.4% received artemether/lumefantrine, quinine or dihydroartemisinin-piperaquine correctly for malaria. Iron and folic supplements were given to nearly 90% of women but often at incorrect doses. CONCLUSIONS: Integrating point-of-care testing into ANC at dispensaries with established HIV testing programmes resulted in a significant increase in testing rates, without disturbing HIV testing rates. While more cases were detected and treated, treatment fidelity still requires strengthening and an integrated monitoring and evaluation system needs to be established.

Treponema pallidum flagellins elicit proinflammatory cytokines from human monocytes via TLR5 signaling pathway.

The tissue damage caused by syphilis infection may be associated with inflammation. However, the virulence factors of Treponema pallidum are still unclear, nor are the molecular mechanisms for leading to the productions of proinflammatory cytokines. Flagellin, a classic pathogen-associated molecular pattern (PAMP), is a potent immunogen that induces inflammation. In the present study, we have demonstrated that stimulations of human monocytes with Treponema pallidum FlaB1, FlaB2, and FlaB3 result in the up regulation of interleukin (IL)-6 and IL-8. Moreover, silencing of the Toll-like receptor 5 (TLR5) gene by using small interfering RNA was found to abrogate the T. pallidum flagellins-induced IL-6 and IL-8 expressions. Similarly, transfection with the dominant negative plasmid encoding MyD88 (pDeNy-hMyD88) was also giving rise to the down regulation of IL-6 and IL-8. We further investigated the relative contributions of mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) signaling to transcriptions and translations of IL-6 and IL-8. Western Blot and immuno fluorescence experiments revealed that flagellins-mediated IL-6 and IL-8 expressions are heavily dependent on ERK, p38, and NF-κB. In addition, inhibitions of p38 kinase, ERK, and NF-κB were found to attenuate the productions of IL-6 and IL-8. Taken together, our results indicate that T. pallidum flagellins can upregulate IL-6 and IL-8 generations via TLR5 and MAPK/NF-κB signaling pathways in THP-1 cells, which will improve our understanding of the pathogenesis of T. pallidum.

Robust pro-inflammatory immune response is associated with serological cure in patients with syphilis: an observational study.

OBJECTIVES: Approximately 15% of adequately treated patients with early syphilis remain serofast. Pathogenesis and clinical significance of this phenomenon is unclear. The objective of this study was to determine whether there is any association between host immune response and treatment outcome (serofast state or proper serological response). METHODS: Forty-four patients with secondary syphilis were enrolled to this study. Levels of pro-inflammatory cytokines such as interferon-γ, tumour necrosis factor-α and interleukin-6 were measured before treatment and 8 hours after injection of antibiotic. RESULTS: After 1 year, based on the serological response patients were stratified into two groups: (1) proper serological response (n=31) and (2) serofast state (n=9). The serological cure rate was 77.5% at 12 months after treatment. Patients with proper serological response had significantly higher levels of analysed cytokines (at baseline and 8 hours after treatment) compared with the serofast state group (p<0.05). CONCLUSIONS: We showed that robust host pro-inflammatory immune response to infection may be the predictive factor of serological cure. The treatment outcome may be also associated with the magnitude of immune reaction occurring during the treatment.

The Tp0684 (MglB-2) Lipoprotein of Treponema pallidum: A Glucose-Binding Protein with Divergent Topology.

Treponema pallidum, the bacterium that causes syphilis, is an obligate human parasite. As such, it must acquire energy, in the form of carbon sources, from the host. There is ample evidence that the principal source of energy for this spirochete is D-glucose acquired from its environment, likely via an ABC transporter. Further, there is genetic evidence of a D-glucose chemotaxis system in T. pallidum. Both of these processes may be dependent on a single lipidated chemoreceptor: Tp0684, also called TpMglB-2 for its sequence homology to MglB of Escherichia coli. To broaden our understanding of this potentially vital protein, we determined a 2.05-Å X-ray crystal structure of a soluble form of the recombinant protein. Like its namesake, TpMglB-2 adopts a bilobed fold that is similar to that of the ligand-binding proteins (LBPs) of other ABC transporters. However, the protein has an unusual, circularly permuted topology. This feature prompted a series of biophysical studies that examined whether the protein's topological distinctiveness affected its putative chemoreceptor functions. Differential scanning fluorimetry and isothermal titration calorimetry were used to confirm that the protein bound D-glucose in a cleft between its two lobes. Additionally, analytical ultracentrifugation was employed to reveal that D-glucose binding is accompanied by a significant conformational change. TpMglB-2 thus appears to be fully functional in vitro, and given the probable central importance of the protein to T. pallidum's physiology, our results have implications for the viability and pathogenicity of this obligate human pathogen.

Treponema pallidum (syphilis) antigen TpF1 induces angiogenesis through the activation of the IL-8 pathway.

Over 10 million people every year become infected by Treponema pallidum and develop syphilis, a disease with broad symptomatology that, due to the difficulty to eradicate the pathogen from the highly vascularized secondary sites of infection, is still treated with injections of penicillin. Unlike most other bacterial pathogens, T. pallidum infection produces indeed a strong angiogenic response whose mechanism of activation, however, remains unknown. Here, we report that one of the major antigen of T. pallidum, the TpF1 protein, has growth factor-like activity on primary cultures of human endothelial cells and activates specific T cells able to promote tissue factor production. The growth factor-like activity is mediated by the secretion of IL-8 but not of VEGF, two known angiogenic factors. The pathogen's factor signals IL-8 secretion through the activation of the CREB/NF-κB signalling pathway. These findings are recapitulated in an animal model, zebrafish, where we observed that TpF1 injection stimulates angiogenesis and IL-8, but not VEGF, secretion. This study suggests that the angiogenic response observed during secondary syphilis is triggered by TpF1 and that pharmacological therapies directed to inhibit IL-8 response in patients should be explored to treat this disease.

Clinicopathologic features, microvessel density, and immunohistochemical expression of ICAM-1 and VEGF in 15 cases of secondary syphilis with oral manifestations.

BACKGROUND: Several studies have reported the clinical aspects of oral syphilis, but few papers have considered the involved immunoinflammatory mechanisms. OBJECTIVE: To analyze the clinicopathologic features and expression of immunoinflammatory and vascular proteins in oral secondary syphilis. STUDY DESIGN: Fifteen patients with oral secondary syphilis were included; histologic features were analyzed using hematoxylin-eosin-stained histologic slides and immunohistochemical reactions were performed against intercellular adhesion molecule 1 (ICAM-1), vascular endothelial growth factor (VEGF), and CD34. RESULTS: Lesions presented as multiple plaques and ulcers mostly located on the tongue, lips, and palate. They were covered by a stratified epithelium of irregular thickness showing exocytosis associated with a subjacent plasma cell infiltrate arranged in perivascular and perineural patterns. ICAM-1 and VEGF were expressed on endothelial cells and leukocytes, and microvessel density was similar on the superficial and deep regions of the connective tissue. CONCLUSIONS: Expression of immunoinflammatory and vascular proteins was intense, suggesting their participation on the pathogenesis of oral secondary syphilis.

[Gene expression profiling of peripheral blood mononuclear cells in patients with secondary syphilis].

OBJECTIVE: To analyze the gene expression profiling characteristics of peripheral blood mononuclear cells (PBMCs) from patients with secondary syphilis, and gain an insight into the host molecular immune mechanisms involved in Treponema pallidum infection. METHODS: Using genome-based high-throughput Illumina sequencing technology, we comprehensively determined the transcriptional difference in PBMCs from 4 secondary syphilis patients and 4 healthy controls, followed by real time PCR to validate the results of Illumina sequencing. RESULTS: Totally, 78 differentially expressed genes were found in the PBMCs of the secondary syphilis patients, among which 16 were associated with the immune system. Significant upregulation was observed in the expressions of pro-inflammatory cytokines and related receptors, such as TNF receptor super-family member 17 (TNFRSF17), IL-17C, IL-21, IL-31 receptor A (IL-31RA), chemokine C-X-C motif ligand 10 (CXCL10), and chemokine (C-C motif) ligand 1 (CCL1), as well as the transcripts for the CD4+ T lymphocytes activation markers CD38, Fc-mediated phagocytosis receptors (FcγR1A, FcγR3B), and complement (C2, SERPING1). CONCLUSIONS: Systemic innate and adaptive immune effecter molecules are involved in the host clearance mechanism of secondary syphilis.

Conservation of the Host-Interacting Proteins Tp0750 and Pallilysin among Treponemes and Restriction of Proteolytic Capacity to Treponema pallidum.

The spirochete Treponema pallidum subsp. pallidum is the causative agent of syphilis, a chronic, sexually transmitted infection characterized by multiple symptomatic and asymptomatic stages. Although several other species in the genus are able to cause or contribute to disease, T. pallidum differs in that it is able to rapidly disseminate via the bloodstream to tissue sites distant from the site of initial infection. It is also the only Treponema species able to cross both the blood-brain and placental barriers. Previously, the T. pallidum proteins, Tp0750 and Tp0751 (also called pallilysin), were shown to degrade host proteins central to blood coagulation and basement membrane integrity, suggesting a role for these proteins in T. pallidum dissemination and tissue invasion. In the present study, we characterized Tp0750 and Tp0751 sequence variation in a diversity of pathogenic and nonpathogenic treponemes. We also determined the proteolytic potential of the orthologs from the less invasive species Treponema denticola and Treponema phagedenis. These analyses showed high levels of sequence similarity among Tp0750 orthologs from pathogenic species. For pallilysin, lower levels of sequence conservation were observed between this protein and orthologs from other treponemes, except for the ortholog from the highly invasive rabbit venereal syphilis-causing Treponema paraluiscuniculi. In vitro host component binding and degradation assays demonstrated that pallilysin and Tp0750 orthologs from the less invasive treponemes tested were not capable of binding or degrading host proteins. The results show that pallilysin and Tp0750 host protein binding and degradative capability is positively correlated with treponemal invasiveness.

Identification of functional candidates amongst hypothetical proteins of Treponema pallidum ssp. pallidum.

Syphilis is a globally occurring venereal disease, and its infection is propagated through sexual contact. The causative agent of syphilis, Treponema pallidum ssp. pallidum, a Gram-negative sphirochaete, is an obligate human parasite. Genome of T. pallidum ssp. pallidum SS14 strain (RefSeq NC_010741.1) encodes 1,027 proteins, of which 444 proteins are known as hypothetical proteins (HPs), i.e., proteins of unknown functions. Here, we performed functional annotation of HPs of T. pallidum ssp. pallidum using various database, domain architecture predictors, protein function annotators and clustering tools. We have analyzed the sequences of 444 HPs of T. pallidum ssp. pallidum and subsequently predicted the function of 207 HPs with a high level of confidence. However, functions of 237 HPs are predicted with less accuracy. We found various enzymes, transporters, binding proteins in the annotated group of HPs that may be possible molecular targets, facilitating for the survival of pathogen. Our comprehensive analysis helps to understand the mechanism of pathogenesis to provide many novel potential therapeutic interventions.

Protective efficacy of a Treponema pallidum Gpd DNA vaccine vectored by chitosan nanoparticles and fused with interleukin-2.

In the present study, immunomodulatory responses of a DNA vaccine constructed by fusing Treponema pallidum (Tp) glycerophosphodiester phosphodiesterase (Gpd) to interleukin-2 (IL-2) and using chitosan (CS) nanoparticles as vectors were investigated. New Zealand white rabbits were immunized by intramuscular inoculation of control DNAs, Tp Gpd DNA vaccine, or Gpd-IL-2 fusion DNA vaccine, which were vectored by CS nanoparticles. Levels of the anti-Gpd antibodies and levels of IL-2 and interferon-γ in rabbits were increased upon inoculation of Gpd-IL-2 fusion DNA vaccine, when compared with the inoculation with Gpd DNA vaccine, with CS vectoring increasing the effects. The Gpd-IL-2 fusion DNA vaccine efficiently enhanced the antigen-specific lymphocyte proliferative response. When the rabbits were challenged intradermally with 10(5) Tp (Nichols) spirochetes, the Gpd-IL-2 fusion DNA vaccine conferred better protection than the Gpd DNA vaccine (P < 0.05), as characterized by lower detectable amounts of dark field positive lesions (17.5%), lower ulcerative lesion scores (15%), and faster recovery. Individuals treated with the Tp Gpd-IL-2 fusion DNA vaccine vectored by CS nanoparticles had the lowest amounts of dark field positive lesions (10%) and ulcerations (5%) observed and the fastest recovery (42 days). These results indicate that the Gpd-IL-2 fusion DNA vaccine vectored by CS nanoparticles can efficiently induce Th1-dominant immune responses, improve protective efficacy against Tp spirochete infection, and effectively attenuate development of syphilitic lesions.

The transition from closed to open conformation of Treponema pallidum outer membrane-associated lipoprotein TP0453 involves membrane sensing and integration by two amphipathic helices.

The molecular architecture and composition of the outer membrane (OM) of Treponema pallidum (Tp), the noncultivable agent of venereal syphilis, differ considerably from those of typical Gram-negative bacteria. Several years ago we described TP0453, the only lipoprotein associated with the inner leaflet of the Tp OM. Whereas polypeptides of other treponemal lipoproteins are hydrophilic, non-lipidated TP0453 can integrate into membranes, a property attributed to its multiple amphipathic helices (AHs). Furthermore, membrane integration of the TP0453 polypeptide was found to increase membrane permeability, suggesting the molecule functions in a porin-like manner. To better understand the mechanism of membrane integration of TP0453 and its physiological role in Tp OM biogenesis, we solved its crystal structure and used mutagenesis to identify membrane insertion elements. The crystal structure of TP0453 consists of an α/ß/α-fold and includes five stably folded AHs. In high concentrations of detergent, TP0453 transitions from a closed to open conformation by lateral movement of two groups of AHs, exposing a large hydrophobic cavity. Triton X-114 phase partitioning, liposome floatation assay, and bis-1-anilino-8-naphthalenesulfonate binding revealed that two adjacent AHs are critical for membrane sensing/integration. Using terbium-dipicolinic acid complex-loaded large unilamellar vesicles, we found that TP0453 increased efflux of fluorophore only at acidic pH. Gel filtration and cross-linking experiments demonstrated that one AH critical for membrane sensing/insertion also forms a dimeric interface. Based on structural dynamics and comparison with Mycobacterium tuberculosis lipoproteins LprG and LppX, we propose that TP0453 functions as a carrier of lipids, glycolipids, and/or derivatives during OM biogenesis.