Platelet-derived major histocompatibility complex class I coating on Treponema pallidum attenuates natural killer cell lethality.

The evasive tactics of Treponema pallidum pose a major challenge in combating and eradicating syphilis. Natural killer (NK) cells mediate important effector functions in the control of pathogenic infection, preferentially eliminating targets with low or no expression of major histocompatibility complex (MHC) class I. To clarify T. pallidum's mechanisms in evading NK-mediated immunosurveillance, experiments were performed to explore the cross-talk relations among T. pallidum, NK cells, and platelets. T. pallidum adhered to, activated, and promoted particle secretion of platelets. After preincubation with T. pallidum, platelets expressed and secreted high levels of MHC class I, subsequently transferring them to the surface of T. pallidum, potentially inducing an immune phenotype characterized by the "pseudo-expression" of MHC class I on the surface of T. pallidum (hereafter referred to a "pseudo-expression" of MHC class I). The polA mRNA assay showed that platelet-preincubated T. pallidum group exhibited a significantly higher copy number of polA transcript than the T. pallidum group. The survival rate of T. pallidum mirrored that of polA mRNA, indicating that preincubation of T. pallidum with platelets attenuated NK cell lethality. Platelets pseudo-expressed the MHC class I ligand on the T. pallidum surface, facilitating binding to killer cell immunoglobulin-like receptors with two immunoglobulin domains and long cytoplasmic tail 3 (KIR2DL3) on NK cells and initiating dephosphorylation of Vav1 and phosphorylation of Crk, ultimately attenuating NK cell lethality. Our findings elucidate the mechanism by which platelets transfer MHC class I to the T. pallidum surface to evade NK cell immune clearance.

Insights into the protective immune response by immunization with full-length recombinant TprK protein: cellular and humoral responses.

Syphilis has resurged in many countries, which has called attention to vaccine development. Based on the immunization-based rabbit model of infection with the Nichols strain, this study explored the protective immune response of a controversial syphilis vaccine candidate, TprK, and found that immunization with full-length rTprK was effective in attenuating lesion development and accelerating lesion resolution, which could reduce the probability of the pathogen spreading to distant tissue sites to prevent the progression of the disease to some extent. Furthermore, the results revealed that immunization with rTprK not only rapidly induced a strong Th1-like cellular response but also elicited a humoral immune response to produce opsonic antibodies to enhance macrophage-mediated opsonophagocytosis. Although complete protection against infection was not achieved, the study provided a comprehensive and in-depth exploration of the immunogenicity of TprK and highlighted the importance of TprK as a promising syphilis vaccine component.

Treponema pallidum promoted microglia apoptosis and prevented itself from clearing by human microglia via blocking autophagic flux.

Treponema pallidum (Tp) has a well-known ability to evade the immune system and can cause neurosyphilis by invading the central nervous system (CNS). Microglia are resident macrophages of the CNS that are essential for host defense against pathogens, this study aims to investigate the interaction between Tp and microglia and the potential mechanism. Here, we found that Tp can exert significant toxic effects on microglia in vivo in Tg (mpeg1: EGFP) transgenic zebrafish embryos. Single-cell RNA sequencing results showed that Tp downregulated autophagy-related genes in human HMC3 microglial cells, which is negatively associated with apoptotic gene expression. Biochemical and cell biology assays further established that Tp inhibits microglial autophagy by interfering with the autophagosome-lysosome fusion process. Transcription factor EB (TFEB) is a master regulator of lysosome biogenesis, Tp activates the mechanistic target of rapamycin complex 1 (mTORC1) signaling to inhibit the nuclear translocation of TFEB, leading to decreased lysosomal biogenesis and accumulated autophagosome. Importantly, the inhibition of autophagosome formation reversed Tp-induced apoptosis and promoted microglial clearance of Tp. Taken together, these findings show that Tp blocks autophagic flux by inhibiting TFEB-mediated lysosomal biosynthesis in human microglia. Autophagosome accumulation was demonstrated to be a key mechanism underlying the effects of Tp in promoting apoptosis and preventing itself from clearing by human microglia. This study offers novel perspectives on the potential mechanism of immune evasion employed by Tp within CNS. The results not only establish the pivotal role of autophagy dysregulation in the detrimental effects of Tp on microglial cells but also bear considerable implications for the development of therapeutic strategies against Tp, specifically involving mTORC1 inhibitors and autophagosome formation inhibitors, in the context of neurosyphilis patients.

The optimum condition of the toluidine red unheated serum test for the replacement of the venereal disease research laboratory test in cerebrospinal fluid for neurosyphilis diagnosis.

Background: The cerebrospinal fluid (CSF) venereal disease research laboratory (VDRL) test remains the standard for the laboratory diagnosis of neurosyphilis. The toluidine red unheated serum test (TRUST) is an alternative to the VDRL test as a serological test for syphilis, but it lacks guidelines for its use in CSF for neurosyphilis diagnosis. Methods: A total of 210 suspected neurosyphilis patients were included, consisting of 124 neurosyphilis patients and 86 syphilis/non-neurosyphilis patients. The TRUST was modified into the CSF-TRUST-10 test with 10 µL of antigen by referring to the CSF-VDRL test, and the CSF-TRUST-17 test with 17 µL of antigen by referring to its procedures in serum. The diagnostic performance of the CSF-TRUST-10 and CSF-TRUST-17 tests and the concordance between them and the CSF-VDRL test were evaluated. Results: The diagnostic performance of the CSF-TRUST-10 and CSF-TRUST-17 tests for diagnosing neurosyphilis were comparable to the CSF-VDRL test, as well as the positive rate. The agreement rate was 98.7% between the qualitative CSF-TRUST-10 and CSF-VDRL tests. A total of 91.4% of the quantitative CSF-TRUST-10 results were consistent with the CSF-VDRL test, and the discordant results were no more than two titres. The agreement rate was 98.1% between the qualitative CSF-TRUST-17 and CSF-VDRL tests and 87.6% between the quantitative CSF-TRUST-17 and CSF-VDRL tests. Conclusions: The CSF-TRUST with 10 µL of antigen could be an alternative for the CSF-VDRL test for neurosyphilis diagnosis. Our results provide a basis for using the TRUST to guide the diagnosis of neurosyphilis.

Longitudinal Variations in the tprK Gene of Treponema pallidum in an Amoy Strain-Infected Rabbit Model.

Heterogeneous tprK sequences have been hypothesized to be an important factor for persistent infection of Treponema pallidum subsp. pallidum (T. pallidum) in humans. Previous research has only explored tprK diversity using a rabbit model infected with almost clonal isolates, which is inconsistent with the fact that infected human isolates contain multiple heterogeneous tprK sequences. Here, we used the T. pallidum Amoy strain with heterogeneous tprK sequences to establish a rabbit infection model and explore longitudinal variations in the tprK gene under normal infection, immunosuppression treatment, and benzathine penicillin G (BPG) treatment using next-generation sequencing. The diversity of the tprK gene was high in all three groups but was highest in the control group and lowest in the BPG group. Interestingly, the overall diversity of tprK in all three groups decreased during infection, exhibiting a "more to less" trend, indicating that survival selection may be an important factor affecting tprK variation in the later infection stage. BPG treatment appeared to reduce the diversity of tprK but increased the frequency of predominant sequence changes, which might facilitate the escape of T. pallidum from the host immune clearance. Furthermore, the original predominant V region sequence did not disappear with disease progression but retained a relatively high proportion within the population, suggesting a new direction for tprK-related vaccine research. This study provides insights into longitudinal variations within the highly heterogeneous tprK gene sequences of T. pallidum and will contribute to further exploration of the pathogenesis of syphilis. IMPORTANCE The tprK variations are an important factor in persistent T. pallidum infection. A nearly clonal isolate has been used previously to investigate the mechanism of tprK gene variations; however, clinical T. pallidum isolates in infected humans exhibit multiple heterogeneous tprK sequences. Here, we use next-generation sequencing to explore longitudinal variations in the tprK gene under normal infection and immunosuppression and benzathine penicillin G treatment in a rabbit model infected with the Amoy strain with heterogeneous tprK sequences. The overall diversity of tprK in all three groups was high and decreased during infection, exhibiting a "more to less" trend. Benzathine penicillin G treatment reduced the diversity of tprK but increased the frequency of predominant sequence changes. Moreover, the original predominant V region sequence did not disappear as the disease progressed but remained at a relatively high proportion within the population. The research results give us a new understanding about tprK variation.

Genetic Profiling of the Full-Length tprK Gene in Patients with Primary and Secondary Syphilis.

TprK antigenic variation is acknowledged as an important strategy developed by Treponema pallidum to achieve immune evasion. Previous studies applied short-read sequencing to explore tprK gene sequence diversity in clinical samples; however, due to the limitations of short-read sequencing, it was difficult to determine the linkage between the seven V regions, and crucial information about full-length tprK variants was lost. Although two recent studies explored complete tprK gene profiles in natural human syphilis infection, there are still too few profiled full-length tprK variants among clinical T. pallidum isolates to fully understand the characteristics of TprK coding diversity. Here, Pacific Biosciences (PacBio) long-read sequencing was applied to examine the diversity of full-length tprK variants in 21 clinical T. pallidum isolates from 11 patients with primary syphilis and 10 patients with secondary syphilis. A total of 398 high-confidence full-length sequences, which presented remarkable sequence heterogeneity, were found. However, these full-length tprK variants exhibited limited variation in length and GC content, showing 24 length types and average GC content of 51.5 ± 0.42% and 51.6 ± 0.26% for primary and secondary syphilis samples, respectively. Additionally, the combined patterns of mutated V regions generating new tprK variants were obviously different in primary and secondary syphilis samples. The diversity of tprK gene sequences in primary syphilis samples may represent the underlying variability of the bacterium; conversely, the variability of the tprK gene in secondary syphilis samples may more accurately reflect how T. pallidum escapes host immune clearance. These data highlight the tprK gene as an important coding gene that shows conflicting genetic characteristics but underlies the persistence of spirochete infection. IMPORTANCE The resurgence of syphilis in both low- and high-income countries has attracted attention, and persistent infection by the pathogen has long been a research focus. The tprK gene, encoding the hypervariable outer membrane protein, is thought to be responsible for pathogen immune evasion and persistent infection. Here, PacBio long-read sequencing was applied to examine the diversity of full-length tprK variants in 21 clinical T. pallidum isolates from 11 patients with primary syphilis and 10 patients with secondary syphilis. The results showed that the sequences of the tprK gene were remarkably heterogeneous; however, the sequences presented limited variation in length and GC content. The investigation of the combined patterns of the V regions allowed us to gain insight into the features of the tprK gene generating new variants at different clinical stages. The findings of this study will be helpful for further exploration of the pathogenesis of syphilis.

False-Positive Results of SARS-CoV-2 RT-PCR in Oropharyngeal Swabs From Vaccinators.

Real-time reverse transcription-polymerase chain reaction (RT-PCR) is the gold standard for diagnosing coronavirus disease 2019 (COVID-19). However, RT-PCR may yield false-positive results, leading to unnecessary countermeasures. Here, we report a "positive" nucleic acid test on a 10-pooled sample during the routine screening that caused many adverse societal effects, and financial and resource losses. However, they were subsequently determined to be a case of vaccine contamination. This case study increases awareness of false-positive RT-PCR results for SARS-CoV-2, especially when participants are vaccinators. Moreover, it could provide relevant suggestions to prevent the recurrence of such incidents.

The whole process of macrophage-Treponema pallidum interactions: Opsonic phagocytosis, nonopsonic phagocytosis and active invasion.

Despite the acknowledged central role of opsonophagocytosis in the process of syphilis, the interaction between Treponema pallidum and human macrophages during nonopsonophagocytosis and active invasion remains controversial. To investigate whether nonopsonic phagocytosis and active invasion, similar to opsonic phagocytosis, also participate in the process of macrophage-T. pallidum interactions, monocyte-derived macrophages were used to study the interactions of T. pallidum and macrophages in the presence of nonsyphytic or syphilitic serum and in the absence of serum in vitro using indirect immunofluorescence and flow cytometry to quantitate treponeme-macrophage interactions. The results showed that macrophages phagocytose T. pallidum under both nonopsonizing conditions (no serum or normal human serum (NHS)) and in the presence of opsonizing serum (secondary syphilitic serum (SSS)) in a time-dependent manner. The percentages of spirochete-positive macrophages in the SSS group were higher than those in the NHS and no-serum groups. Blocking FcγR or inactivating complement caused a significant decrease in the percentage of spirochete-positive macrophages in the SSS group but did not cause a decrease in the percentages of spirochete-positive macrophages in the NHS and no-serum groups. In addition, after inhibiting macrophage phagocytosis, approximately 30% of macrophages internalized spirochetes, verifying that T. pallidum actively penetrated macrophages rather than was ingested by them. This study provides evidence that opsonic phagocytosis, nonopsonic phagocytosis and active invasion are all active during T. pallidum-macrophage interactions and reveals a process of treponeme-macrophage interactions in T. pallidum pathogenesis.

LncRNA-ENST00000421645 Upregulates Kank1 to Inhibit IFN-γ Expression and Promote T Cell Apoptosis in Neurosyphilis.

Long non-coding RNAs are involved in many infectious diseases. Our previous studies showed that lncRNA-ENST00000421645 expression is increased in T lymphocytes of neurosyphilis patients compared to healthy controls. However, whether lncRNA-ENST00000421645 has biological functions remains unclear. The current study was undertaken to understand the mechanism of lncRNA-ENST00000421645 in T lymphocyte function in neurosyphilis patients. The lncRNA-ENST00000421645 pull-down assay showed that lncRNA-ENST00000421645 acted on the acetylase NAT10. The chromatin immunoprecipitation (ChIP)-PCR results showed that lncRNA-ENST00000421645 promoted the acetylation of histone H3K27 adjacent to the Kank1 promoter, thereby promoting Kank1 protein expression. Kank1 promotes 14-3-3 protein expression, inhibits NF-kB activation, inhibits IFN-γ secretion by T lymphocytes, and promotes T lymphocyte apoptosis. Taken together, our findings suggest a novel mechanism that LncRNA-ENST00000421645 upregulates Kank1 to inhibit IFN-γ expression and promote T cell apoptosis in neurosyphilis.

Identification of Treponema pallidum-specific protein biomarkers in syphilis patient serum using mass spectrometry.

Aim: To screen novel biomarkers in serum of syphilis patients using a mass spectrometry-based method. Materials & methods: Sera were collected from 18 syphilis patients and divided into three groups. Every six serum samples (before and after treatment) in each group were pooled and detected by mass spectrometry. Results: Twenty-five unique peptides corresponding to 15 Treponema pallidum proteins were discovered. Among them, Tp0369 was discovered as a promising biomarker candidate in this study. Tp0524 and Tp0984 levels decreased 0.38-fold and 0.51-fold after BPG treatment, respectively, which may be related to disease outcomes of syphilis. Conclusion: These findings confirmed the presence of detectable T. pallidum protein in patients' serum, which could promote the development of syphilis diagnostics.

LncRNA-ENST00000421645 promotes T cells to secrete IFN-γ by sponging PCM1 in neurosyphilis.

Aim: Neurosyphilis patients exhibited significant expression of long noncoding RNA (lncRNA) in peripheral blood T lymphocytes. In this study, we further clarified the role of lncRNA-ENST00000421645 in the pathogenic mechanism of neurosyphilis. Methods: lncRNA-ENST00000421645 was transfected into Jurkat-E6-1 cells, namely lentivirus (Lv)-1645 cells. RNA pull-down assay, flow cytometry, RT-qPCR, ELISA (Neobioscience Technology Co Ltd, Shenzhen, China) and RNA immunoprecipitation chip assay were used to analyze the function of lncRNA-ENST00000421645. Results: The expression of IFN-γ in Lv-1645 cells was significantly increased compared to that in Jurkat-E6-1 cells stimulated by phorbol-12-myristate-13-acetate (PMA). Then, it was suggested that lncRNA-ENST00000421645 interacts with PCM1 protein. Silencing PCM1 significantly increased the level of IFN-γ in Lv-1645 cells stimulated by PMA. Conclusion: This study revealed that lncRNA-ENST00000421645 mediates the production of IFN-γ by sponging PCM1 protein after PMA stimulation.

Lay abstract The mechanisms underlying Treponema pallidum (a type of bacterium that causes syphilis) invasion into the CNS have not yet been established. In this study, we further clarified the role of long noncoding RNA (lncRNA) in the pathogenic process causing nerve damage. The results suggested that lncRNA-ENST00000421645 interacts with an important protein named PCM1. Suppressing the expression of PCM1 significantly increased the level of IFN-γ cytokines (substances secreted by immune cells that effect other cells) with an increased level of lncRNA-ENST00000421645 when immune cells were stimulated by phorbol-12-myristate-13-acetate a specific activator of the PKC signaling enzyme involved in gene transcription pathways. This study revealed that lncRNA-ENST00000421645 mediates the production of IFN-γ by interacting with PCM1 protein.

Characterisation of the novel clinical isolate X-4 containing a new tp0548 sequence-type.

OBJECTIVES: A novel tp0548 sequence-type was identified in one clinical isolate (X-4) from a patient diagnosed with primary syphilis in Xiamen, China. To precisely define and characterise a new clinical isolate, we performed further genome-scale molecular analysis. METHODS: The pooled segment genome sequencing method followed by Illumina sequencing was performed. RESULTS: This novel sequence-type contained a unique nucleotide substitution 'T' at position 167 and belonged to the SS14-like clade of TPA strains, as determined by phylogenetic analysis. Multi-locus sequence analysis of nine chromosomal loci demonstrated that the X-4 isolate was clustered within a monophyletic group of TPA strains. Whole-genome phylogenetic analysis subsequently corroborated the TPA strain classification of the X-4 isolate and revealed that the isolate was closely related to the SS14 strain, with 42 single-nucleotide variations and 12 insertions/deletions. In addition, high intrastrain heterogeneity in the length of the poly G/C tracts was found in the TPAChi_0347 locus, which might indicate that this gene of the X-4 isolate is likely involved in phase variation events. The length heterogeneity of the poly A/T tracts was lower than the genetic variability of the poly G/C tracts, and all the observed intrastrain variations fell within coding regions. CONCLUSION: The novel tp0548 sequence-type was determined to belong to a new TPA isolate, X-4. The identification of variable length in homopolymetic tracts (G/C and A/T) could provide a snapshot of the genes that potentially involved in genotype-phenotype variations. These findings provide an unequivocal characterisation for better understanding the molecular variation of this emerging isolate.

Treponemapallidum Dysregulates Monocytes and Promotes the Expression of IL-1ß and Migration in Monocytes Through the mTOR Signaling Pathway.

Monocytes are widely involved in the body's defense response, and abnormally regulated monocyte subsets are closely related to the pathogenesis of various diseases. It is unclear whether Treponema pallidum (Tp) dysregulates monocyte subsets and impacts the functions of monocytes. This study aims to analyze the distribution of monocyte subsets in syphilis patients and the effect of Tp on monocyte functions to explore the pathogenesis of syphilis. Flow cytometry was employed to detect monocyte subsets. With or without pre-treatment with rapamycin, THP-1 cell migration stimulated by Tp was investigated by a Transwell migration assay, and THP-1 cell phagocytosis was studied using fluorescent microspheres. IL-1ß and TNF-α expression was quantified by PCR and flow cytometry, while LC3 and mTOR were investigated in Tp-exposed THP-1 cells using western blotting. Tp infection led to an increase in the proportion of CD14++CD16+ monocytes and a decrease in the proportion of CD14++CD16- monocytes. In addition, Tp promoted monocyte (THP-1) CD14 and CD16 expression in vitro, induced the expression of IL-1ß and TNF-α in a dose-dependent manner and promoted the migration and autophagy of monocytes. Furthermore, mTOR phosphorylation on monocytes was stimulated by Tp, and the levels peaked at 30 min. Pre-treatment with rapamycin (mTOR inhibitor) attenuated the expression of IL-1ß and migration in Tp-exposed THP-1 cells. Tp abnormally regulates monocyte subsets and promotes migration, autophagy, and the expression of IL-1ß and TNF-α in THP-1 cells. Meanwhile, the mTOR affected the expression of IL-1ß and migration in Tp-exposed THP-1 cells. This study is important as it sheds light on the mechanism by which monocytes interact with Tp during infection.

Immunization with nontreponemal antigen alters the course of experimental syphilis in the rabbit model.

The role of nontreponemal antibodies in the Treponema pallidum infection course is unclear. We investigated the effect of immunization with nontreponemal antigen on T. pallidum-challenged rabbits. Nontreponemal antigen was injected intravenously into rabbits in the nontreponemal group (n = 12) to elicit antibodies (≥1:64), and normal saline-injected rabbits were used as controls (n = 12). Then, rabbits were challenged with 106T. pallidum per site along their back. Lesion development was observed, and the injection sites were biopsied for mRNA analysis every week. Six rabbits from both groups were euthanized at 14 d and 28 d. The popliteal lymph nodes were extracted to assess infectivity using a rabbit infectivity test. The maximum lesion diameters were not different between the two groups (12.4 ± 0.9 mm in the nontreponemal group vs. 12.5 ± 1.0 mm in the control group, P = 0.386), but the time to maximum diameter appearance was delayed by approximately 4 d in the nontreponemal group (14.4 ± 1.6 d vs. 10.8 ± 1.9 d, P = 0.000). There were no significant differences in the proportions of lesions (58/60 (96.7%) vs. 59/60 (98.3%), P = 0.500) or ulcers (55/60 (91.7%) vs. 57/60 (95.0%), P = 0.359) between the two groups. An ulcer development delay of 5 d was observed in the nontreponemal group (19.3 ± 2.0 d vs. 14.0 ± 1.8 d, P = 0.000). IL-2 and IFN-γ mRNA expression in the nontreponemal group was significantly higher than that in the control group at 7 d and 14 d post-challenge. flaA mRNA expression and the rabbit infectivity test positive rate were not different between the two groups. Immunization with nontreponemal antigen altered the syphilis course in rabbits, resulting in delayed maximal lesion diameter and ulcer development, but it could not inhibit the spread of T. pallidum from primary lesion sites to viscera.

Tp0136 targets fibronectin (RGD)/Integrin ß1 interactions promoting human microvascular endothelial cell migration.

Lesion healing without treatment is a unique clinical characteristic of the early stages of syphilis infection. Angiogenesis, which involves endothelial cell migration, is an important process in wound healing. Tp0136, an outer membrane protein of T. pallidum, has the ability to bind host fibronectin-producing cells, which plays a crucial role in the pathogenesis of syphilis. In this research, we purposed to analyze the role of Tp0136 in the migration of human microvascular endothelial (HMEC-1) cells and to explore the related mechanism. First, Tp0136 significantly promoted HMEC-1 cell migration. Furthermore, the levels of C-C motif ligand 2 (CCL2) mRNA and protein expression rose with the concentration and time increasing of Tp0136. The migration of HMEC-1 cells was significantly suppressed by an anti-CCL2 antibody and a CCR2 (the CCL2 receptor) inhibitor. Further study revealed that, in cells pretreated with anti-fibronectin antibody, anti-integrin ß1 antibody or RGD (Arg-Gly-Asp), the expression levels of CCL2 induced by Tp0136 were notably decreased. Additionally, after pretreatment with an anti-fibronectin antibody, an anti-integrin ß1 antibody or RGD, the migration of HMEC-1 cells treated with Tp0136 was obviously suppressed. These results show that Tp0136 promots the migration of HMEC-1 cells by inducing CCL2 expression via the interaction of the fibronectin RGD domain with integrin ß1 and the CCL2/CCR2 signaling pathway, and these interactions may contribute to the mechanisms that increase the capacity for self-healing syphilis infection.

Effect of anti-TP0136 antibodies on the progression of lesions in an infected rabbit model.

The effect of anti-TP0136 antibodies on the progression of syphilis is poorly understood. This study aimed to investigate the effect of anti-TP0136 antibodies on the progression of lesions in an infected rabbit model. Intramuscular injection of rTP0136 into rabbits in the immunized group (n = 4) elicited high titers of anti-TP0136 antibodies, and rabbits were then challenged with 105T. pallidum per site along their back. Lesion development was observed, and the injection sites were biopsied for tp0574 mRNA and histological analyses every week until the wound healed. The rabbits in the control group were injected with normal saline instead of rTP0136. Viable T. pallidum in the challenged rabbits was assessed with rabbit infectivity tests. The lesions in the immunized group took longer to heal than those in the control group (42 d vs. 28 d, P < 0.001) and had markedly higher levels of total cellular infiltrates. The mRNA level of tp0574 in the immunized group was significantly higher than that in the control group (P < 0.05). Viable T. pallidum was detected in rabbit lymph nodes in both the immunized and control groups. Our study showed that high titers of anti-TP0136 antibodies promoted the infiltration of inflammatory cells into local lesions and intensified tissue damage, thus delaying wound healing, and had no protective effect on the occurrence of syphilis in the rabbit model.

The P2X7 receptor mediates NLRP3-dependent IL-1ß secretion and promotes phagocytosis in the macrophage response to Treponema pallidum.

It is unclear whether P2X7 receptor (P2X7R) mediates NOD-like receptor family protein 3 (NLRP3)-dependent IL-1ß secretion and spirochete phagocytosis in syphilis. This study was conducted to investigate the role of P2X7R in modifying NLRP3-dependent IL-1ß secretion and regulating phagocytosis by Treponema pallidum (T. pallidum)-induced macrophages. Macrophages derived from a human acute monocytic leukemia cell line were cultured with T. pallidum. The activation of P2X7R in T. pallidum-treated macrophages occurred in a dose- and time-dependent manner. The P2X7R silencing group showed significantly decreased NLRP3 mRNA and protein levels (vs. the Tp group, P < 0.001). Similar results were observed for IL-1ß secretion using ELISA (vs. the Tp group, P < 0.001). Furthermore, P2X7R siRNA transfection significantly decreased the percentage of spirochete-positive macrophages (29.73% vs. 70.83%, P < 0.001) and spirochete internalization (mean fluorescence intensity (MFI), 9.20 vs. 19.39, P < 0.001). This finding revealed that P2X7R played a role in the induction of NLRP3-dependent IL-1ß secretion by T. pallidum-induced macrophages. Furthermore, we found that P2X7R plays an important role in IL-1ß secretion and in the promotion of T. pallidum phagocytosis by macrophages. These results may not only contribute to our understanding of the immune mechanism that is active during T. pallidum infection but may also lay the groundwork for strategies to combat syphilis.

Treponema pallidum Induces the Secretion of HDVSMC Inflammatory Cytokines to Promote the Migration and Adhesion of THP-1 Cells.

The pathological features of syphilis, a disease caused by Treponema pallidum (T. pallidum), are characterized by vascular involvement with endarteritis and periarteritis. Little is known about the interactions of infiltrating immunocytes with human dermal vascular smooth muscle cells (HDVSMCs) in arterioles during the immunopathogenesis of syphilis. In the present study, we demonstrated that stimulation of HDVSMCs with T. pallidum resulted in the upregulated gene transcription and protein expression of interleukin (IL)-6, monocyte chemoattractant protein-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1) in a dose- and time-dependent manner. Moreover, 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 T. pallidum activated the NF-κB signaling pathway in HDVSMCs. Inhibition of NF-κB suppressed T. pallidum-induced IL-6, MCP-1, and ICAM-1 expression. In addition, the migration and adhesion of THP-1 cells to T. pallidum-treated HDVSMCs were significantly decreased by pretreatment with an NF-κB inhibitor. These findings demonstrate that T. pallidum induces the production of IL-6, MCP-1, and ICAM-1 in HDVSMCs and promotes the adherence and migration of THP-1 cells to HDVSMCs through the NF-κB signaling pathway, which may provide new insight into the pathogenesis of T. pallidum infection.

Insights into the genetic variation profile of tprK in Treponema pallidum during the development of natural human syphilis infection.

BACKGROUND: Although the tprK gene of Treponema pallidum are thought to play a critical role in the pathogenesis of syphilis, the profile of variations in tprK during the development of human syphilis infection have remained unclear. METHODS/PRINCIPAL FINDINGS: Through next-generation sequencing, we compared the tprK gene of 14 secondary syphilis patients with that of 14 primary syphilis patients, and the results showed an increased number of variants within the seven V regions of the tprK gene in the secondary syphilis samples. The length of the sequences within each V region also presented a 3-bp changing pattern. Interestingly, the frequencies of predominant sequences within the V regions in the secondary syphilis samples were generally decreased compared with those found in the primary syphilis samples, particularly in the V7 region, where a frequency below 60% was found in up to 57% (8/14) of all secondary samples compared with 7% (1/14) of all primary samples. Moreover, the number of minor variants distributed between frequencies of 10 and 49.9% was increased. The alignment of all amino acid sequences within each V region of the primary and secondary syphilis samples revealed that some amino acid sequences, particularly the amino acid sequences IASDGGAIKH and IASEDGSAGNLKH in V1, were highly stable. Additionally, the amino acid sequences in V6 also exhibited notable intrastrain heterogeneity and were likely to form a strain-specific pattern at the interstrain level. CONCLUSIONS: The identification of different profiles of the tprK gene in primary and secondary syphilis patients indicated that the tprK gene of T. pallidum undergoes constant variation to result in the best adaptation to the host. The highly stable peptides found in V1 are likely promising potential vaccine components. The highly heterogenetic regions (e.g., V6) could help to understand the role of tprK in immune evasion.

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.