Treponema pallidum subsp. pallidum with an Artificially impaired TprK antigenic variation system is attenuated in the Rabbit model of syphilis.

BACKGROUND: The TprK protein of the syphilis agent, Treponema pallidum subsp. pallidum (T. pallidum), undergoes antigenic variation in seven discrete variable (V) regions via non-reciprocal segmental gene conversion. These recombination events transfer information from a repertoire of 53 silent chromosomal donor cassettes (DCs) into the single tprK expression site to continually generate TprK variants. Several lines of research developed over the last two decades support the theory that this mechanism is central to T. pallidum's ability for immune avoidance and persistence in the host. Structural and modeling data, for example, identify TprK as an integral outer membrane porin with the V regions exposed on the pathogen's surface. Furthermore, infection-induced antibodies preferentially target the V regions rather than the predicted ß-barrel scaffolding, and sequence variation abrogates the binding of antibodies elicited by antigenically different V regions. Here, we engineered a T. pallidum strain to impair its ability to vary TprK and assessed its virulence in the rabbit model of syphilis. PRINCIPAL FINDINGS: A suicide vector was transformed into the wild-type (WT) SS14 T. pallidum isolate to eliminate 96% of its tprK DCs. The resulting SS14-DCKO strain exhibited an in vitro growth rate identical to the untransformed strain, supporting that the elimination of the DCs did not affect strain viability in absence of immune pressure. In rabbits injected intradermally with the SS14-DCKO strain, generation of new TprK sequences was impaired, and the animals developed attenuated lesions with a significantly reduced treponemal burden compared to control animals. During infection, clearance of V region variants originally in the inoculum mirrored the generation of antibodies to these variants, although no new variants were generated in the SS14-DCKO strain to overcome immune pressure. Naïve rabbits that received lymph node extracts from animals infected with the SS14-DCKO strain remained uninfected. CONCLUSION: These data further support the critical role of TprK in T. pallidum virulence and persistence during infection.

Treponema pallidum Periplasmic and Membrane Proteins Are Recognized by Circulating and Skin CD4+ T Cells.

BACKGROUND: Histologic and serologic studies suggest the induction of local and systemic Treponema pallidum-specific CD4+ T-cell responses to T. pallidum infection. We hypothesized that T. pallidum-specific CD4+ T cells are detectable in blood and in the skin rash of secondary syphilis and persist in both compartments after treatment. METHODS: Peripheral blood mononuclear cells collected from 67 participants were screened by interferon-γ (IFN-γ) ELISPOT response to T. pallidum sonicate. T. pallidum-reactive T-cell lines from blood and skin were probed for responses to 89 recombinant T. pallidum antigens. Peptide epitopes and HLA class II restriction were defined for selected antigens. RESULTS: We detected CD4+ T-cell responses to T. pallidum sonicate ex vivo. Using T. pallidum-reactive T-cell lines we observed recognition of 14 discrete proteins, 13 of which localize to bacterial membranes or the periplasmic space. After therapy, T. pallidum-specific T cells persisted for at least 6 months in skin and 10 years in blood. CONCLUSIONS: T. pallidum infection elicits an antigen-specific CD4+ T-cell response in blood and skin. T. pallidum-specific CD4+ T cells persist as memory in both compartments long after curative therapy. The T. pallidum antigenic targets we identified may be high-priority vaccine candidates.

In Vitro Isolation of Treponema pallidum subsp. pallidum From Fresh and Frozen Needle Aspirates of Primary Experimental Syphilis Lesions.

ABSTRACT: Isolation of Treponema pallidum subsp. pallidum strains still relies on rabbit intratesticular inoculation of clinical samples. In this article, we report an alternative isolation approach based on the inoculation of fresh and frozen needle aspirates of primary experimental lesions into culture plates suitable for in vitro propagation of the syphilis agent.

Previous Syphilis Alters the Course of Subsequent Episodes of Syphilis.

BACKGROUND: The influence of previous syphilis on the course of a subsequent episode is unknown. METHODS: Individuals enrolled in a study of cerebrospinal fluid (CSF) abnormalities in syphilis were allowed to enroll in the study again with subsequent syphilis. For each participant, the index episode was defined as the most recent syphilis episode for which the study entry visit was performed within 30 days of the syphilis diagnosis date. Venipuncture and lumbar puncture (LP) were performed. Total number of syphilis episodes was determined by review of medical and public health records. T. pallidum DNA in blood and rRNA in CSF were detected by polymerase chain reaction (PCR) and reverse transcriptase PCR. Odds ratios (ORs) with 95% confidence intervals (95% CI) were determined by logistic regression. RESULTS: 651 individuals had one (n = 482), two (n = 121) or three or more (n = 48) episodes of syphilis. The proportion of individuals whose index episode was early latent stage was significantly higher in those with ≥3 syphilis episodes; this relationship was reduced to a trend when rate of testing was taken into account. Adjusted odds (aOR) of detection of T. pallidum DNA in blood or rRNA in CSF at the index episode were significantly lower in those with previous syphilis (0.17 [95% CI, 0.09-0.31] and 0.15 [95% CI, 0.07-0.35]). The aOR for neurosyphilis at the index episode was also significantly lower in individuals with previous syphilis (0.54 [95% CI, 0.34-0.87]). CONCLUSIONS: Previous syphilis attenuates the manifestations of subsequent infection with T. pallidum.

A Comparison of Treponema pallidum Subspecies pallidum Molecular Typing Systems: Multilocus Sequence Typing vs. Enhanced Centers for Disease Control and Prevention Typing.

BACKGROUND: At least 3 syphilis typing systems are proposed. Recent work suggests that multilocus sequence typing (MLST) may be superior to enhanced Centers for Disease Control and Prevention typing (ECDCT) by yielding a higher discriminatory power. The goal of this study was to compare the 2 systems and identify associations between neurosyphilis and strain types. METHODS: Multilocus sequence typing for tp0136, tp0548, and tp0705 was determined for DNA from 78 Treponema pallidum subspecies pallidum isolates propagated in rabbits, 10 oral and 10 genital or non-genital lesion swabs, and 10 blood samples from patients with syphilis. These samples were chosen because they were completely typeable by ECDCT. Using both systems, association between strain types and neurosyphilis, defined as a reactive cerebrospinal fluid Venereal Disease Research Laboratory test, was determined. Partial and complete ECDCT types were also determined for samples from different anatomical sites in 35 patients, and from blood and blood isolates (rabbit propagated) from 13 patients. RESULTS: The MLST type could be fully determined for 100 (92.6%) of 108 samples. Although MLST subdivided 3 common ECDCT types, it failed to distinguish among others. Neurosyphilis was more common in individuals infected with type 1.1.2 and tp0705 type 2 using MLST, and tp0548 type f using ECDCT. Enhanced Centers for Disease Control and Prevention typing was stable among anatomical sites and between patient-derived and rabbit propagated organisms. CONCLUSIONS: Compared with ECDCT, MLST was not uniformly more discriminating. Both typing systems demonstrate that specific types may be more neurotropic than others.

Detection of Treponema pallidum DNA in Oropharyngeal Swabs and Whole Blood for Syphilis Diagnosis.

BACKGROUND: Syphilis diagnosis relies on serological tests, which may be falsely nonreactive or may be reactive but not reflect current syphilis. METHODS: Polymerase chain reaction for detection of T. pallidum DNA was performed on 123 oropharyngeal swabs, 120 whole bloods, and 46 lesion exudate swabs from 123 untreated individuals with syphilis (cases); oropharyngeal swabs from 148 at-risk controls without syphilis; and 73 oropharyngeal swabs and 36 whole bloods from 73 individuals recently treated for syphilis. RESULTS: Most (90.2%) cases had early syphilis. T. pallidum DNA was detected in 33 (26.8%) of 123 oropharyngeal swabs, 32 (26.7%) of 120 bloods, and 30 (65.2%) of 46 lesion exudate swabs. T. pallidum DNA was detected in 49 (40.8%) of 120 individuals in whom both oropharyngeal swabs and blood were tested. T. pallidum was more likely to be amplified from oropharyngeal swabs when it was amplified from blood than when it was not (15 of 32 [46.9%] vs. 17 of 88 [19.3%], P = 0.003). For each 2-fold increase in serum rapid plasma reagin titer, the odds of detection of T. pallidum DNA in oropharyngeal swabs increased by 1.44 (95% confidence interval, 1.14-1.82, P = 0.003). T. pallidum DNA was not detected in oropharyngeal samples from controls, but it was detected in 3 (8.3%) of 36 bloods from individuals recently treated for syphilis: 2 at 1 day and 1 at 5 days after initiation of syphilis treatment. CONCLUSIONS: Nucleic amplification tests can identify recent T. pallidum infection and may be particularly useful for diagnosis of very early or asymptomatic syphilis.

A Dual-Platform Point-of-Care Test for Neurosyphilis Diagnosis.

BACKGROUND: The diagnosis of neurosyphilis relies on cerebrospinal fluid (CSF) abnormalities (pleocytosis, elevated protein) and CSF-Venereal Disease Research Laboratory (VDRL) test. In resource-limited settings, the CSF-VDRL test may not be widely available. METHODS: We optimized a commercial immunochromatographic strip test, the DPP Chembio syphilis assay, for performance with CSF and tested centrifuged CSF samples of 71 patients with syphilis (35 with neurosyphilis and 36 without neurosyphilis). A CSF dilution of 1:4 was chosen based on agreement with CSF pools with documented results from the CSF-VDRL test and fluorescent treponemal antibody absorption test on CSF. Using an electronic reader, we obtained unit values of treponemal and nontreponemal antibodies for all study samples and generated a receiver operating characteristic curve; using the Youden index, we established diagnostic cutoffs with optimal sensitivity and specificity. RESULTS: Diagnostic sensitivity of the nontreponemal test was 80% (95% confidence interval, 63%-92%) and specificity was 97% (95% confidence interval, 85%-100%) for neurosyphilis diagnosis using a reactive CSF-VDRL that improved after neurosyphilis therapy as a criterion standard. CONCLUSIONS: In this small study, the DPP Chembio test showed promising results for neurosyphilis diagnosis. Further studies are needed to assess its performance in resource-limited settings.

Neurosyphilis Treatment Outcomes After Intravenous Penicillin G Versus Intramuscular Procaine Penicillin Plus Oral Probenecid.

BACKGROUND: Data comparing neurosyphilis treatment regimens are limited. METHODS: Participants were enrolled in a study of cerebrospinal fluid (CSF) abnormalities in syphilis that was conducted at the University of Washington between April 2003 to May 2014. They were diagnosed with syphilis and referred by their providers due to concerns for neurosyphilis. We evaluated 150 people with CSF abnormalities who were treated with either intravenous aqueous penicillin G (PenG) or intramuscular aqueous procaine penicillin G plus oral probenecid (APPG-P). An abnormal CSF diagnosis was defined as a white blood cell (WBC) count >20/µL, a CSF protein reading >50 mg/dL, or a reactive CSF-Venereal Disease Research Laboratory test (VDRL). Hazard ratios for normalization of CSF or serum measures were determined using Cox regression. RESULTS: In individuals treated with either PenG or APPG-P, CSF WBCs and CSF-VDRL reactivity normalized within 12 months after treatment, while protein normalized more slowly and less completely. There was no relationship between treatment regimen or human immunodeficiency virus (HIV) status and likelihood of normalization of any measure. Among those living with HIV, CSF WBC counts and CSF-VDRL reactivity were more likely to normalize in those treated with antiretrovirals. Unexpectedly, CSF WBCs were more likely to normalize in those with low CD4+ T cell counts. When neurosyphilis was more stringently defined as a reactive CSF-VDRL, the relationship with the CD4+ T cell count remained unchanged. CONCLUSIONS: In the current antiretroviral treatment era, neurosyphilis treatment outcomes are not different for PenG and APPG-P, regardless of HIV status. The relationship between the normalization of CSF WBC counts and CD4+ T cell counts may indicate continued imprecision in neurosyphilis diagnostic criteria, due to HIV-related CSF pleocytosis.

Previous Syphilis Alters the Course of Subsequent Episodes of Syphilis.

BACKGROUND: Individuals with previous syphilis may be more likely to be asymptomatic when they are reinfected with Treponema pallidum. METHODS: Individuals enrolled in a study of cerebrospinal fluid (CSF) abnormalities in syphilis were allowed to enroll in the study again with subsequent syphilis. For each participant, the index episode was defined as the most recent syphilis episode for which the study entry visit was performed within 30 days of the syphilis diagnosis date. Venipuncture and lumbar puncture were performed. The total number of syphilis episodes was determined by review of medical and public health records. Treponema pallidum DNA in blood and rRNA in CSF were detected using polymerase chain reaction (PCR) and reverse transcriptase PCR. Odds ratios (ORs) with 95% confidence intervals (CIs) were determined using logistic regression. RESULTS: 701 individuals had 1 (n = 478), 2 (n = 155), or ≥3 (n = 68) episodes of syphilis. The proportion of individuals whose index episode was asymptomatic significantly increased with increased number of syphilis episodes (P < .001). This difference was not explained by frequency of serological tests. Adjusted ORs (aORs) of detection of T. pallidum DNA in blood or rRNA in CSF at the index episode were significantly lower in those with previous syphilis (0.13; 95% CI, .08-.23, and 0.06, 95% CI, .02-.17). The aOR of neurosyphilis at the index episode was also significantly lower in individuals with previous syphilis (0.43; 95% CI, .27-.68). CONCLUSIONS: Previous syphilis attenuates clinical and laboratory manifestations of infection with T. pallidum.

Neurosyphilis Increases Human Immunodeficiency Virus (HIV)-associated Central Nervous System Inflammation but Does Not Explain Cognitive Impairment in HIV-infected Individuals With Syphilis.

BACKGROUND: Individuals infected with human immunodeficiency virus (HIV) who have previously had syphilis may have cognitive impairment. We tested the hypothesis that neurosyphilis causes cognitive impairment in HIV by amplifying HIV-related central nervous system (CNS) inflammation. METHODS: HIV-infected participants enrolled in a study of cerebrospinal fluid (CSF) abnormalities in syphilis underwent the mental alternation test (MAT), venipuncture, and lumbar puncture. CSF concentrations of chemokine (C-X-C motif) ligand 10 (CXCL10), chemokine (C-C motif) ligand 2 (CCL2), and neurofilament light (NFL) were determined by commercial assays. The proportion of peripheral blood mononuclear cells (PBMCs) and of CSF white blood cells (WBCs) that were activated monocytes (CD14+CD16+) was determined by flow cytometry. Neurosyphilis was defined as detection of Treponema pallidum 16S RNA in CSF or CSF white blood cells (WBCs) >20/uL or a reactive CSF-Venereal Disease Research Laboratory (VDRL) test; uncomplicated syphilis was defined as undetectable CSF T. pallidum, CSF WBCs ≤5/uL and nonreactive CSF-VDRL. MAT <18 was considered low. RESULTS: Median proportion of PBMCs that were activated monocytes (16.6 vs. 5.3), and median CSF CXCL10 (10658 vs. 2530 units), CCL2 (519 vs. 337 units) and HIV RNA (727 vs. 50 c/mL) were higher in neurosyphilis than in uncomplicated syphilis (P ≤ .001 for all comparisons). Neurosyphilis was not related to low MAT scores. Participants with low MAT scores had higher median CSF CXCL10 (10299 vs. 3650 units, P = .008) and CCL2 (519 vs. 365 units, P = .04) concentrations than those with high MAT scores. CONCLUSIONS: Neurosyphilis may augment HIV-associated CNS inflammation, but it does not explain cognitive impairment in HIV-infected individuals with syphilis.

Cerebrospinal Fluid Treponema pallidum Particle Agglutination Assay for Neurosyphilis Diagnosis.

Limited data suggest that the cerebrospinal fluid Treponema pallidum particle agglutination assay (CSF-TPPA) is sensitive and a CSF Treponema pallidum hemagglutination assay (CSF-TPHA) titer of ≥1:640 is specific for neurosyphilis diagnosis. CSF-TPPA reactivity and titer were determined for a convenience sample of 191 CSF samples from individuals enrolled in a study of CSF abnormalities in syphilis (training data set). The sensitivity of a reactive test and the specificity for reactivity at serial higher CSF dilutions were determined. Subsequently, CSF-TPPA reactivity at a 1:640 dilution was determined for all available samples from study participants enrolled after the last training sample was collected (validation data set, n = 380). Neurosyphilis was defined as (i) a reactive CSF Venereal Disease Research Laboratory test (CSF-VDRL), (ii) detection of T. pallidum in CSF by reverse transcriptase PCR, or (iii) new vision loss or hearing loss. In the training data set, the diagnostic sensitivities of a reactive CSF fluorescent treponemal antibody absorption test (CSF-FTA-ABS) and a reactive CSF-TPPA did not differ significantly (67 to 98% versus 76 to 95%). The specificity of a CSF-TPPA titer of ≥1:640 was significantly higher than that of lower dilutions and was not significantly different from that of CSF-VDRL. In the validation data set, the diagnostic specificity of a CSF-TPPA titer of ≥1:640 was high and did not differ significantly from that of CSF-VDRL (93 to 94% versus 90 to 91%). Ten CSF samples with a nonreactive CSF-VDRL had a CSF-TPPA titer of ≥1:640. If a CSF-TPPA titer of ≥1:640 was used in addition to a reactive CSF-VDRL, the number of neurosyphilis diagnoses would have increased from 47 to 57 (21.3%). A CSF-TPPA titer cutoff of ≥1:640 may be useful in identifying patients with neurosyphilis when CSF-VDRL is nonreactive.

Reduced Treponema pallidum-Specific Opsonic Antibody Activity in HIV-Infected Patients With Syphilis.

BACKGROUND: Human immunodeficiency virus (HIV)-infected individuals may have poorer serological responses to syphilis treatment and may be more likely to experience neurosyphilis. Treponema pallidum is cleared from sites of infection by opsonization, ingestion, and killing by macrophages. METHODS: Serum samples from 235 individuals with syphilis were tested for T. pallidum-specific opsonic activity. Blood T. pallidum concentrations were determined by real-time polymerase chain reaction amplification of the tp0574 gene, and T. pallidum was detected in cerebrospinal fluid (CSF) by reverse-transcriptase polymerase chain reaction of 16S ribosomal RNA. RESULTS: Opsonic activity was higher with higher serum rapid plasma reagin titers (P < .001), and in those treated for uncomplicated syphilis before serum collection (P < .001). Opsonic activity was lower in HIV-infected than in HIV-uninfected individuals even after the above factors were taken into account (P = .006). In participants in whom blood T. pallidum was detectable, those with the highest opsonic activity had lower blood T. pallidum concentrations. In multivariable analyses, there was not a significant relationship between opsonic activity and detection of T. pallidum in CSF or CSF-VDRL reactivity. CONCLUSIONS: Serum T. pallidum-specific opsonic activity is significantly lower in HIV-infected individuals. Impaired T. pallidum-specific immune responses could contribute to differences in the course of disease or treatment response.

Molecular Typing of Treponema pallidum in Ocular Syphilis.

BACKGROUND: Syphilis can have many clinical manifestations, including eye involvement, or "ocular syphilis." In 2015, an increase in reported cases of ocular syphilis and potential case clusters raised concern for an oculotropic strain of Treponema pallidum, the infectious agent of syphilis. Molecular typing was used to examine strains found in cases of ocular syphilis in the United States. METHODS: In 2015, after a clinical advisory issued by the Centers for Disease Control and Prevention, pretreatment clinical specimens from US patients with ocular syphilis were sent to a research laboratory for molecular analysis of T. pallidum DNA. Molecular typing was conducted on these specimens, and results were compared with samples collected from Seattle patients diagnosed with syphilis, but without ocular symptoms. RESULTS: Samples were typed from 18 patients with ocular syphilis and from 45 patients with syphilis, but without ocular symptoms. Clinical data were available for 14 ocular syphilis patients: most were men, human immunodeficiency virus-infected, and had early syphilis. At least 5 distinct strain types of Treponema pallidum were identified in these patients, and 9 types were identified in the Seattle nonocular patients. 14d/g was the most common type in both groups. An unusual strain type was detected in a small cluster of ocular syphilis patients in Seattle. CONCLUSIONS: Ocular syphilis is a serious sequela of syphilis. In this preliminary study, clear evidence of a predominant oculotropic strain causing ocular syphilis was not detected. Identification of cases and prompt treatment is critical in the management of ocular syphilis.

Macrolide Resistance in Treponema pallidum Correlates With 23S rDNA Mutations in Recently Isolated Clinical Strains.

BACKGROUND: High rates of 23S rDNA mutations implicated in macrolide resistance have been identified in Treponema pallidum samples from syphilis patients in many countries. Nonetheless, some clinicians have been reluctant to abandon azithromycin as a treatment for syphilis, citing the lack of a causal association between these mutations and clinical evidence of drug resistance. Although azithromycin resistance has been demonstrated in vivo for the historical Street 14 strain, no recent T. pallidum isolates have been tested. We used the well-established rabbit model of syphilis to determine the in vivo efficacy of azithromycin against 23S rDNA mutant strains collected in 2004 to 2005 from patients with syphilis in Seattle, Wash. METHODS: Groups of 9 rabbits were each infected with a strain containing 23S rDNA mutation A2058G (strains UW074B, UW189B, UW391B) or A2059G (strains UW228B, UW254B, and UW330B), or with 1 wild type strain (Chicago, Bal 3, and Mexico A). After documentation of infection, 3 animals per strain were treated with azithromycin, 3 were treated with benzathine penicillin G, and 3 served as untreated control groups. Treatment efficacy was documented by darkfield microscopic evidence of T. pallidum, serological response, and rabbit infectivity test. RESULTS: Azithromycin uniformly failed to cure rabbits infected with strains harboring either 23S rDNA mutation, although benzathine penicillin G was effective. Infections caused by wild type strains were successfully treated by either azithromycin or benzathine penicillin G. CONCLUSIONS: A macrolide resistant phenotype was demonstrated for all strains harboring a 23S rDNA mutation, demonstrating that either A2058G or A2059G mutation confers in vivo drug resistance.

Point-of-care treponemal tests for neurosyphilis diagnosis.

BACKGROUND: The laboratory diagnosis of neurosyphilis rests upon identifying cerebrospinal fluid (CSF) abnormalities, including CSF-Venereal Disease Research Laboratory (VDRL) reactivity. The CSF-VDRL may not be available in the parts of the world where neurosyphilis is most common. Treponemal immunochromatographic strip tests (ICSTs) have been developed as point-of-care tests on blood for syphilis diagnosis in resource-limited settings. METHODS: We optimized 3 commercial ICSTs for performance on CSF and tested CSF samples from 217 patients with syphilis. The Syphicheck-WB test (Qualpro Diagnostics, Goa, India; "Syphicheck") was chosen for further study based on agreement with CSF-VDRL test results. We determined CSF-Syphicheck titers for 152 samples. We modified the CSF-Syphicheck for point-of-care testing in a US sexually transmitted diseases clinic and compared results on 102 paired centrifuged and uncentrifuged CSF samples obtained in the laboratory to the results obtained at point of care; results of samples diluted 1:4 were compared in a subset. RESULTS: The diagnostic sensitivity of a reactive CSF-Syphicheck (62%-64%) and the diagnostic specificity of a CSF-Syphicheck titer at or above 1:4 (79%-81%) were equivalent to the CSF-VDRL (54%-69% sensitivity, 73%-75% specificity) for laboratory and clinical neurosyphilis diagnoses. The CSF-Syphicheck normalized after neurosyphilis therapy similarly to the CSF-VDRL. The modified CSF-Syphicheck performed well at the point of care, albeit with better performance on cell-free compared with uncentrifuged CSF. CONCLUSIONS: Cerebrospinal fluid treponemal ICSTs hold promise for point-of-care neurosyphilis diagnosis in regions where the CSF-VDRL is not available. Further study should address the performance of CSF ICSTs in resource-limited settings.

Toll-like receptor polymorphisms are associated with increased neurosyphilis risk.

BACKGROUND: Single-nucleotide polymorphisms (SNPs) in toll-like receptors (TLR) 1, 2, and 6 impair cell signaling in response to spirochetal lipoproteins. We investigated whether common SNPs in TLR1, TLR2, or TLR6 were associated with laboratory- or clinically-defined neurosyphilis. METHODS: Polymorphisms in the genes for TLR1 (a T→G mutation at position 1805), TLR2 (a G→A mutation at position 2258), and TLR6 (a C→T mutation at position 745) were sought in 456 white patients with syphilis. Laboratory-defined neurosyphilis included a reactive cerebrospinal fluid (CSF)-Venereal Disease Research Laboratory (VDRL) test. Clinically-defined neurosyphilis included new vision or hearing loss. Controls had CSF white blood cells of 5/µL or less, nonreactive CSF-Venereal Disease Research Laboratory, and no vision or hearing loss. RESULTS: Overall, 26.2% of patients had laboratory-defined and 36.2% had clinically-defined neurosyphilis. Compared with controls, patients with any of the 3 SNPs were more likely to have laboratory-defined neurosyphilis. Those with TLR2 or TLR6 SNPs were more likely to have clinically-defined neurosyphilis. These associations were independent of serum rapid plasma reagin titer. CONCLUSIONS: A common TLR1 polymorphism is associated with an increased risk of laboratory-defined neurosyphilis, and common TLR2 and TLR6 polymorphisms are associated with an increased risk of both laboratory- and clinically-defined neurosyphilis. These data suggest that host factors impact the natural history of syphilis.

Bright New Resources for Syphilis Research: Genetically Encoded Fluorescent Tags for Treponema pallidum and Sf1Ep Cells.

The recently discovered methodologies to cultivate and genetically manipulate Treponema pallidum subsp. pallidum ( T. pallidum ) have significantly helped syphilis research, allowing the in vitro evaluation of antibiotic efficacy, performance of controlled studies to assess differential treponemal gene expression, and generation of loss-of-function mutants to evaluate the contribution of specific genetic loci to T. pallidum virulence. Building on this progress, we engineered the T. pallidum SS14 strain to express a red-shifted Green Fluorescent Protein (GFP) and Sf1Ep cells to express mCherry and blue fluorescent protein (BFP) for enhanced visualization. These new resources improve microscopy- and cell sorting-based applications for T. pallidum , better capturing the physical interaction between the host and pathogen, among other possibilities. Continued efforts to develop and share new tools and resources are required to help our overall knowledge of T. pallidum biology and syphilis pathogenesis reach that of other bacterial pathogens, including spirochetes. Graphical abstract: By employing genetic engineering, T. pallidum was modified to express GFP, and Sf1Ep cells to express mCherry on the cytoplasmic membrane and BFP in the nucleus. These new resources for syphilis research will facilitate experimental designs to better define the complex interplay between T. pallidum and the host during infection.

Treponema pallidum periplasmic and membrane proteins are recognized by circulating and skin CD4+ T cells.

Background: Histologic and serologic studies suggest the induction of local and systemic Treponema pallidum ( Tp )-specific CD4+ T cell responses to Tp infection. We hypothesized that Tp -specific CD4+ T cells are detectable in blood and in the skin rash of secondary syphilis and persist in both compartments after treatment. Methods: PBMC collected from 67 participants were screened by IFNγ ELISPOT response to Tp sonicate. Tp -reactive T cell lines from blood and skin were probed for responses to 88 recombinant Tp antigens. Peptide epitopes and HLA class II restriction were defined for selected antigens. Results: We detected CD4+ T cell responses to Tp sonicate ex vivo. Using Tp -reactive T cell lines we observed recognition of 14 discrete proteins, 13 of which localize to bacterial membranes or the periplasmic space. After therapy, Tp -specific T cells persisted for at least 6 months in skin and 10 years in blood. Conclusions: Tp infection elicits an antigen-specific CD4+ T cell response in blood and skin. Tp -specific CD4+ T cells persist as memory in both compartments long after curative therapy. The Tp antigenic targets we identified may be high priority vaccine candidates.

Treponema pallidum subsp. pallidum with an Artificially Impaired TprK Antigenic Variation System is Attenuated in the Rabbit Model of Syphilis.

Background: The TprK protein of the syphilis agent, Treponema pallidum subsp. pallidum ( T. pallidum ), undergoes antigenic variation in seven discrete variable (V) regions via non-reciprocal segmental gene conversion. These recombination events transfer information from a repertoire of 53 silent chromosomal donor cassettes (DCs) into the single tprK expression site to continually generate TprK variants. Several lines of research developed over the last two decades support the theory that this mechanism is central to T. pallidum 's ability for immune avoidance and persistence in the host. Structural and modeling data, for example, identify TprK as an integral outer membrane porin with the V regions exposed on the pathogen's surface. Furthermore, infection-induced antibodies preferentially target the V regions rather than the predicted ß-barrel scaffolding, and sequence variation abrogates the binding of antibodies elicited by antigenically different V regions. Here, we engineered a T. pallidum strain to impair its ability to vary TprK and assessed its virulence in the rabbit model of syphilis. Principal findings: A suicide vector was transformed into the wild-type (WT) SS14 T. pallidum isolate to eliminate 96% of its tprK DCs. The resulting SS14-DC KO strain exhibited an in vitro growth rate identical to the untransformed strain, supporting that the elimination of the DCs did not affect strain viability in absence of immune pressure. In rabbits injected intradermally with the SS14-DC KO strain, generation of new TprK sequences was impaired, and the animals developed attenuated lesions with a significantly reduced treponemal burden compared to control animals. During infection, clearance of V region variants originally in the inoculum mirrored the generation of antibodies to these variants, although no new variants were generated in the SS14-DC KO strain to overcome immune pressure. Naïve rabbits that received lymph node extracts from animals infected with the SS14-DC KO strain remained uninfected. Conclusion: These data further support the critical role of TprK in T. pallidum virulence and persistence during infection. Author Summary: Syphilis is still endemic in low- and middle-income countries, and it has been resurgent in high-income nations, including the U.S., for years. In endemic areas, there is still significant morbidity and mortality associated with this disease, particularly when its causative agent, the spirochete Treponema pallidum subsp . pallidum ( T. pallidum ) infects the fetus during pregnancy. Improving our understanding of syphilis pathogenesis and T. pallidum biology could help investigators devise better control strategies for this serious infection. Now that tools to genetically manipulate this pathogen are available, we can engineer T. pallidum strains lacking specific genes or genomic regions known (or believed) to be associated with virulence. This approach can shed light on the role of the ablated genes or sequences in disease development using loss-of-function strains. Here, we derived a knockout (KO) T. pallidum mutant (SS14-DC KO ) impaired in its ability to undergo antigenic variation of TprK, a protein that has long been hypothesized to be central in evasion of the host immune response and pathogen persistence during infection. When compared to the WT isolate, which is still capable of antigenic variation, the SS14-DC KO strain is significantly attenuated in its ability to proliferate and to induce early disease manifestations in infected rabbits. Our results further support the importance of TprK antigenic variation in syphilis pathogenesis and pathogen persistence.