Clinical presentation of early syphilis and genomic sequences of Treponema pallidum strains in patient specimens and isolates obtained by rabbit inoculation.

BACKGROUND: The global resurgence of syphilis necessitates vaccine development. METHODS: We collected ulcer exudates and blood from 17 primary syphilis (PS) participants and skin biopsies and blood from 51 secondary syphilis (SS) participants in Guangzhou, China for Treponema pallidum subsp. pallidum (TPA) qPCR, whole genome sequencing (WGS), and isolation of TPA in rabbits. RESULTS: TPA DNA was detected in 15 of 17 ulcer exudates and 3 of 17 blood PS specimens. TPA DNA was detected in 50 of 51 SS skin biopsies and 27 of 51 blood specimens. TPA was isolated from 47 rabbits with success rates of 71% (12/17) and 69% (35/51), respectively, from ulcer exudates and SS bloods. We obtained paired genomic sequences from 24 clinical samples and corresponding rabbit isolates. Six SS14- and two Nichols-clade genome pairs contained rare discordances. Forty-one of the 51 unique TPA genomes clustered within SS14 subgroups largely from East Asia, while 10 fell into Nichols C and E subgroups. CONCLUSIONS: Our TPA detection rate was high from PS ulcer exudates and SS skin biopsies and over 50% from SS blood, with TPA isolation in over two-thirds of samples. Our results support the use of WGS from rabbit isolates to inform vaccine development.

The incidence of new cases of syphilis has skyrocketed globally in the twenty-first century. This global resurgence requires new strategies, including vaccine development. As part of an NIH funded Cooperative Research Center to develop a syphilis vaccine, we established a clinical research site in Guangzhou, China to better define the local syphilis epidemic and obtain samples from patients with primary and secondary syphilis for whole genome sequencing (WGS) of circulating Treponema pallidum strains. Inoculation of rabbits enabled us to obtain T. pallidum genomic sequences from spirochetes disseminating in blood, a compartment of immense importance for syphilis pathogenesis. Collectively, our results further clarify the molecular epidemiology of syphilis in southern China, enrich our understanding of the manifestations of early syphilis, and demonstrate that the genomic sequences of spirochetes obtained by rabbit inoculation accurately represent those of the spirochetes infecting the corresponding patients.

Uncovering the mechanisms of host mitochondrial cardiolipin release in syphilis: Insights from human microvascular endothelial cells.

The release of host mitochondrial cardiolipin is believed to be the main factor that contributes to the production of anti-cardiolipin antibodies in syphilis. However, the precise mechanism by which mitochondria release cardiolipin in this context remains elusive. This study aimed to elucidate the mechanisms underlying mitochondrial cardiolipin release in syphilis. We conducted a cardiolipin quantitative assay and immunofluorescence analysis to detect mitochondrial cardiolipin release in human microvascular endothelial cells (HMEC-1), with and without Treponema pallidum (Tp) infection. Furthermore, we explored apoptosis, a key mechanism for mitochondrial cardiolipin release. The potential mediator molecules were then analyzed through RNA-sequence and subsequently validated using in vitro knockout techniques mediated by CRISPR-Cas9 and pathway-specific inhibitors. Our findings confirm that live-Tp is capable of initiating the release of mitochondrial cardiolipin, whereas inactivated-Tp does not exhibit this capability. Additionally, apoptosis detection further supports the notion that the release of mitochondrial cardiolipin occurs independently of apoptosis. The RNA-sequencing results indicated that microtubule-associated protein2 (MAP2), an axonogenesis and dendrite development gene, was up-regulated in HMEC-1 treated with Tp, which was further confirmed in syphilitic lesions by immunofluorescence. Notably, genetic knockout of MAP2 inhibited Tp-induced mitochondrial cardiolipin release in HMEC-1. Mechanically, Tp-infection regulated MAP2 expression via the MEK-ERK-HES1 pathway, and MEK/ERK phosphorylation inhibitors effectively block Tp-induced mitochondrial cardiolipin release. This study demonstrated that the infection of live-Tp enhanced the expression of MAP2 via the MEK-ERK-HES1 pathway, thereby contributing to our understanding of the role of anti-cardiolipin antibodies in the diagnosis of syphilis.

Transcriptome-wide assessment of N6-methyladenosine modification identifies different gene expression and infection-associated pathways in Treponema pallidum-infected macrophage.

BACKGROUND: Treponema pallidum (Tp) is a widespread and destructive pathogen that leads to syphilis. As the acknowledged executor of host immunity, macrophage plays vital roles in combating the invasion and migration of Tp. However, the mechanisms of these processes are largely unknown, especially the critical driver genes and associated modifications. OBJECTIVE: We aimed to systematically dissect the global N6-methyladenosine (m6A) RNA modification patterns in Tp-infected macrophages. METHODS: The RNA of Tp-infected/non-infected macrophage was extracted, followed by mRNA sequencing and methylated RNA immunoprecipitation (MeRIP) sequencing. Bioinformatics analysis was executed by m6A peaks and motifs identification, Gene ontology and signaling pathways analysis of differentially expressed genes, and comprehensive comparison. The m6A levels were measured by RNA Methylation Assay, and m6A modified genes were determined by qPCR. RESULTS: Totally, 2623 unique and 3509 common m6A peaks were proved along with related transcripts in Tp-infected macrophages. The common m6A-related genes were enriched in the signals of oxidative stress, cell differentiation, and angiogenesis, while unique genes in those of metabolism, inflammation, and infection. And differentially expressed transcripts revealed various biological processes and pathways associated with catabolic and infection. They also experienced comprehensive analysis due to hyper-/hypo-methylation. And the m6A level of macrophage was elevated, along with qPCR validation of specific genes. CONCLUSION: With a particular m6A transcriptome-wide map, our study provides unprecedented insights into the RNA modification of macrophage stimulated by Tp in vitro, which partially differs from other infections and may provide clues to explore the immune process for syphilis.

Virulence and Adhesion of the Treponema pallidum Nichols Strain Simultaneously Decrease in a Continuous-Infection New Zealand White Rabbit Model.

Syphilis is a sexually transmitted disease caused by T. pallidum, and the T. pallidum Nichols strain is widely used with the New Zealand white rabbit model for evaluating drug and vaccine protection. However, changes in the virulence of T. pallidum during transmission are still unknown. Herein, we explored the virulence of T. pallidum in the rabbit model of continuous infection through phenotype observation and further investigated the relationship between virulence and adhesion. During the construction of the syphilis rabbit model, the optimal dose of 104/site of T. pallidum was determined to effectively observe the depiction of syphilis lesions and immune responses for further virulence evaluation. Its virulence was gradually weakened during the interaction with host cells or the testicular passage, which was also proven using the pathological phenotype of the syphilis rabbit model. In addition, the adhesive ability of T. pallidum was reduced with increasing generation, which was verified via the co-incubation of the pathogen with Sf1Ep cells. This study provides insight into the relationship by which the virulence and adhesion of T. pallidum were decreased in a New Zealand white rabbit model of continuous infection and contributes to our knowledge regarding the development of syphilis.

Early syphilis in Guangzhou, China: presentation, molecular detection of Treponema pallidum , and genomic sequences in clinical specimens and isolates obtained by rabbit infectivity testing.

Background: The global resurgence of syphilis requires novel prevention strategies. Whole genome sequencing (WGS) of Treponema pallidum ( TPA ) using different specimen types is essential for vaccine development. Methods: Patients with primary (PS) and secondary (SS) syphilis were recruited in Guangzhou, China. We collected ulcer exudates and blood from PS participants, and skin biopsies and blood from SS participants for TPA polA polymerase chain reaction (PCR); ulcer exudates and blood were also used to isolate TPA strains by rabbit infectivity testing (RIT). TPA WGS was performed on 52 ulcer exudates and biopsy specimens and 25 matched rabbit isolates. Results: We enrolled 18 PS and 51 SS participants from December 2019 to March 2022. Among PS participants, TPA DNA was detected in 16 (89%) ulcer exudates and three (17%) blood specimens. Among SS participants, TPA DNA was detected in 50 (98%) skin biopsies and 27 (53%) blood specimens. TP A was isolated from 48 rabbits, with a 71% (12/17) success rate from ulcer exudates and 69% (36/52) from SS bloods. Twenty-three matched SS14 clade genomes were virtually identical, while two Nichols clade pairs had discordant tprK sequences. Forty-two of 52 unique TPA genomes clustered in an SS14 East Asia subgroup, while ten fell into two East Asian Nichols subgroups. Conclusions: Our TPA detection rate was high from PS ulcer exudates and SS skin biopsies and over 50% from SS whole blood, with RIT isolation in over two-thirds of samples. Our results support the use of WGS from rabbit isolates to inform vaccine development. Summary: We performed Treponema pallidum molecular detection and genome sequencing from multiple specimens collected from early syphilis patients and isolates obtained by rabbit inoculation. Our results support the use of whole genome sequencing from rabbit isolates to inform syphilis vaccine development.

Clinical and genomic diversity of Treponema pallidum subsp. pallidum: A global, multi-center study of early syphilis to inform vaccine research.

Background: The continuing increase in syphilis rates worldwide necessitates development of a vaccine with global efficacy. We conducted a multi-center, observational study to explore Treponema pallidum subsp. pallidum ( TPA ) molecular epidemiology essential for vaccine research by analyzing clinical data and specimens from early syphilis patients using whole-genome sequencing (WGS) and publicly available WGS data. Methods: We enrolled patients with primary (PS), secondary (SS) or early latent (ELS) syphilis from clinics in China, Colombia, Malawi and the United States between November 2019 - May 2022. Inclusion criteria included age ≥18 years, and syphilis confirmation by direct detection methods and/or serological testing. TPA detection and WGS were conducted on lesion swabs, skin biopsies/scrapings, whole blood, and/or rabbit-passaged isolates. We compared our WGS data to publicly available genomes, and analysed TPA populations to identify mutations associated with lineage and geography. Findings: We screened 2,820 patients and enrolled 233 participants - 77 (33%) with PS, 154 (66%) with SS, and two (1%) with ELS. Median age of participants was 28; 66% were cis -gender male, of which 43% reported identifying as "gay", "bisexual", or "other sexuality". Among all participants, 56 (24%) had HIV co-infection. WGS data from 113 participants demonstrated a predominance of SS14-lineage strains with geographic clustering. Phylogenomic analysis confirmed that Nichols-lineage strains are more genetically diverse than SS14-lineage strains and cluster into more distinct subclades. Differences in single nucleotide variants (SNVs) were evident by TPA lineage and geography. Mapping of highly differentiated SNVs to three-dimensional protein models demonstrated population-specific substitutions, some in outer membrane proteins (OMPs) of interest. Interpretation: Our study involving participants from four countries substantiates the global diversity of TPA strains. Additional analyses to explore TPA OMP variability within strains will be vital for vaccine development and improved understanding of syphilis pathogenesis on a population level. Funding: National Institutes of Health, Bill and Melinda Gates Foundation.

YTHDF1 Negatively Regulates Treponema pallidum-Induced Inflammation in THP-1 Macrophages by Promoting SOCS3 Translation in an m6A-Dependent Manner.

Background: Previous studies have confirmed that the bacterium Treponema pallidum (TP) or its proteins provide signals to macrophages that induce an inflammatory response; however, little is known about the negative regulation of this macrophage-mediated inflammatory response during syphilis infection or the underlying mechanism. Recent evidence suggests the role of the RNA modification, N6-adenosine methylation (m6A), in regulating the inflammatory response and pathogen-host cell interactions. Therefore, we hypothesized that m6A plays a role in the regulation of the inflammatory response in macrophages exposed to TP. Methods: We first assessed m6A levels in TP-infected macrophages differentiated from the human monocyte cell line THP-1. The binding and interaction between the m6A "writer" methyltransferase-like 3 (METTL3) or the m6A "reader" YT521-B homology (YTH) domain-containing protein YTHDF1 and the suppressor of cytokine signaling 3 (SOCS3), as a major regulator of the inflammatory response, were explored in differentiated TP-infected THP-1 cells as well as in secondary syphilitic lesions from patients. The mechanisms by which YTHDF1 and SOCS3 regulate the inflammatory response in macrophages were assessed. Results and Conclusion: After macrophages were stimulated by TP, YTHDF1 was upregulated in the cells. YTHDF1 was also upregulated in the syphilitic lesions compared to adjacent tissue in patients. YTHDF1 recognizes and binds to the m6A methylation site of SOCS3 mRNA, consequently promoting its translation, thereby inhibiting the JAK2/STAT3 pathway, and reducing the secretion of inflammatory factors, which results in anti-inflammatory regulation. This study provides the first demonstration of the role of m6A methylation in the pathological process of syphilis and further offers new insight into the pathogenesis of TP infection.

A suite of PCR-LwCas13a assays for detection and genotyping of Treponema pallidum in clinical samples.

The performance of commonly used assays for diagnosis of syphilis varies considerably depending on stage of infection and sample type. In response to the need for improved syphilis diagnostics, we develop assays that pair PCR pre-amplification of the tpp47 gene of Treponema pallidum subsp. pallidum with CRISPR-LwCas13a. The PCR-LwCas13a assay achieves an order of magnitude better analytical sensitivity than real-time PCR with equivalent specificity. When applied to a panel of 216 biological specimens, including 135 clinically confirmed primary and secondary syphilis samples, the PCR-LwCas13a assay demonstrates 93.3% clinical sensitivity and 100% specificity, outperforming tpp47 real-time PCR and rabbit-infectivity testing. We further adapt this approach to distinguish Treponema pallidum subsp. pallidum lineages and identify genetic markers of macrolide resistance. Our study demonstrates the potential of CRISPR-based approaches to improve diagnosis and epidemiological surveillance of syphilis.

Desmoglein 2 (DSG2) Is A Receptor of Human Adenovirus Type 55 Causing Adult Severe Community-Acquired Pneumonia.

Human adenovirus type 55 (HAdV-B55) is a re-emergent acute respiratory disease pathogen that causes adult community-acquired pneumonia (CAP). Previous studies have shown that the receptor of HAdV-B14, which genome is highly similar with HAdV-B55, is human Desmoglein 2 (DSG2). However, whether the receptor of HAdV-B55 is DSG2 is undetermined because there are three amino acid mutations in the fiber gene between HAdV-B14 and HAdV-B55. Here, firstly we found the 3T3 cells, a mouse embryo fibroblast rodent cell line which does not express human DSG2, were able to be infected by HAdV-B55 after transfected with pcDNA3.1-DSG2, while normal 3T3 cells were still unsusceptible to HAdV-B55 infection. Next, A549 cells with hDSG2 knock-down by siRNA were hard to be infected by HAdV-B3/-B14/-B55, while the control siRNA group was still able to be infected by all these types of HAdVs. Finally, immunofluorescence confocal microscopy indicated visually that Cy3-conjugated HAdV-B55 viruses entered A549 cells by binding to DSG2 protein. Therefore, DSG2 is a major receptor of HAdV-B55 causing adult CAP. Our finding is important for better understanding of interactions between adenoviruses and host cells and may shed light on the development of new drugs that can interfere with these processes as well as for the development of potent prophylactic vaccines.