Search

Background Treponema pallidum (T. pallidum) is transmitted from person to person by direct contact with widespread dissemination early after acquisition. Current syphilis diagnostic testing is limited to darkfield microscopy from visible lesions and serologic antibody detection. T. pallidum molecular diagnostics are urgently needed. Methods As part of a multicenter US syphilis treatment trial that completed enrollment in March 2022 (NCT 3637660), we randomized 249 adults with early syphilis infection to 1 vs 3 weekly doses of benzathine penicillin G to compare treatment response at 6 months. In a small substudy (n=32) presented here, consenting participants at the University of Alabama at Birmingham (UAB) and Emory University had pre-treatment oral (buccal mucosa and posterior pharynx) and lesion swab samples collected. Following DNA extraction, T. pallidum burden in samples was quantified by targeting the tp0574 gene. Syphilis was staged by experienced providers according to physical examination and serology. Results Study participants were men (100%) and most were living with HIV (91%) with mean CD4 count 477 cells/mm3. Syphilis stage was categorized as primary in 6 (19%), secondary in 18 (56%) and early latent in 8 (25%). Oral swab qPCR positivity rates were 17% in primary disease, 44% in secondary disease, and 63% in early latent disease (see Figure). Lesion PCR positivity was similar in primary and secondary syphilis (40% and 38.5%). Mean treponemal burden was highest on oral swabs of participants with early latent syphilis (13,128,969) and lesion swabs of participants with secondary syphilis (2,033,309). Conclusion Among adult males, many living with HIV, data suggest a role for oral and lesion swabs for the molecular detection of T. pallidum. Widespread availability of PCR, the relative ease of oral specimen collection, and the opportunity to directly demonstrate T. pallidum in persons with early latent syphilis suggest the opportunity for PCR testing to enhance and refine syphilis diagnostic testing. The elevated proportion of positive oral samples and significant organism burden in secondary and early latent syphilis is consistent with potential for transmission. Disclosures All Authors: No reported disclosures.

Sequencing of most Treponema pallidum (T. pallidum) genomes excludes repeat regions in tp0470 and the tp0433 gene, encoding the acidic repeat protein (arp). As a first step to understanding the evolution and function of these genes and the proteins they encode, we developed a protocol to nanopore sequence tp0470 and arp genes from 212 clinical samples collected from ten countries on six continents. Both tp0470 and arp repeat structures recapitulate the whole genome phylogeny, with subclade-specific patterns emerging. The number of tp0470 repeats is on average appears to be higher in Nichols-like clade strains than in SS14-like clade strains. Consistent with previous studies, we found that 14-repeat arp sequences predominate across both major clades, but the combination and order of repeat type varies among subclades, with many arp sequence variants limited to a single subclade. Although strains that were closely related by whole genome sequencing frequently had the same arp repeat length, this was not always the case. Structural modelling of TP0470 suggested that the eight residue repeats form an extended α-helix, predicted to be periplasmic. Modeling of the ARP revealed a C-terminal sporulation-related repeat (SPOR) domain, predicted to bind denuded peptidoglycan, with repeat regions possibly incorporated into a highly charged β- sheet. Outside of the repeats, all TP0470 and ARP amino acid sequences were identical. Together, our data, along with functional considerations, suggests that both TP0470 and ARP proteins may be involved in T. pallidum cell envelope remodeling and homeostasis, with their highly plastic repeat regions playing as-yet-undetermined roles.

Although the isolation of Treponema pallidum subsp. pallidum (T. pallidum) from a syphilis patient dates to 1912, for the duration of the 20

Despite more than a century of research, genetic manipulation of Treponema pallidum subsp. pallidum (T. pallidum), the causative agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding of the mechanisms behind T. pallidum success as a pathogen. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible to experiment with transformation and selection protocols in this pathogen. Here, we describe an approach that successfully replaced the tprA (tp0009) pseudogene in the SS14 T. pallidum strain with a kanamycin resistance (kanR) cassette. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kanR gene was cloned downstream of the tp0574 gene promoter. The tp0574prom-kanR cassette was then placed between two 1-kbp homology arms identical to the sequences upstream and downstream of the tprA pseudogene. To induce homologous recombination and integration of the kanR cassette into the T. pallidum chromosome, in vitro-cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl2-based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kanR cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole-genome sequencing (WGS) of transformed treponemes propagated in vitro and/or in vivo. ddPCR analysis of RNA and mass spectrometry confirmed expression of the kanR message and protein in treponemes propagated in vitro. Moreover, tprA knockout (tprAko-SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin. We demonstrated that genetic manipulation of T. pallidum is attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development., Author summary Syphilis is still an endemic disease in many low- and middle-income countries, and it has been resurgent in high-income nations for almost two decades. In endemic areas, syphilis causes significant morbidity and mortality, particularly when its causative agent, the spirochete Treponema pallidum subsp. pallidum (T. pallidum) is transmitted to the fetus during pregnancy. A better understanding of T. pallidum biology and syphilis pathogenesis would help devise better control strategies for this infection. One of the limitations associated with working with T. pallidum was our inability to genetically alter this pathogen to evaluate the function of genes encoding virulence factors or create attenuated strains that could be informative for vaccine development when studied using the rabbit model of the disease. Here, we report a transformation protocol that allowed us to replace a specific region of the T. pallidum genome containing a pseudogene (i.e., a non-functional gene) with a stably integrated kanamycin resistance gene. To our knowledge, this is the first-ever report of a method to achieve a genetically modified T. pallidum strain.

Background Syphilis, caused by the spirochete Treponema pallidum subsp. pallidum (T. pallidum), continues to be a significant global health concern. The diagnosis of syphilis mainly relies on assessing clinical manifestations and performing serologic testing to detect treponemal and non-treponemal antibodies to discriminate between past and present infection and to evaluate response to treatment. Limitations, however, include that no test can discriminate among syphilis stages, and sensitivity is low during the very early stages of infection. New diagnostics could circumvent some of these deficiencies. Methods To this end, we developed a novel array of T. pallidum recombinant proteins to identify seroreactive proteins and compare their performance to that of antigens commonly used in treponemal tests such as the 17 and 47 kDa lipoproteins (Tp0435 and Tp0574, respectively). Antigens were applied to the array, then reactivity was assessed via indirect enzyme-linked immunosorbent assay (ELISA). For this analysis, we used sera samples from 124 patients collected at various timepoints (0 months, 3 months, and 6 months). Samples from all three timepoints were available from 29 patients. Results The control antigens demonstrated the highest reactivity against the sera. Among the antigens tested so far Tp0954, a putative adhesin of the syphilis agent, exhibited seroreactivity similar to that of the controls. Conclusion Our data support Tp0954 as a possible candidate to improve the performance of currently available treponemal tests.

Background The recently described method for in vitro cultivation of the syphilis agent, Treponema pallidum subsp. pallidum (T. pallidum), paved the way to transformation experiments to genetically engineer this pathogen. The demonstration that genetically modified syphilis treponemes are attainable would represent a milestone in syphilis research, destined to revolutionize our approach to understand the mechanisms behind T. pallidum success as a pathogen. Here, for the first time in syphilis research, we describe a transformation protocol that successfully replaced the tprA (tp0009) pseudogene in the SS14 T. pallidum strain with a kanamycin resistance (kanR) cassette. Principal findings A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kanR gene was cloned downstream of the tp0574 gene promoter. The tp0574prom-kanR cassette was then placed between two 1-Kbp homology arms identical to the sequences upstream and downstream of the tprA pseudogene. To induce homologous recombination of the arms into the T. pallidum chromosome, with resulting integration of the kanR cassette, in vitro-cultured SS14 strain spirochetes were exposed to the engineered vector resuspended in a transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kanR cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR) and whole genome sequencing (WGS) of transformant treponemes propagated in vitro and in vivo. ddPCR analysis of RNA and mass spectrometry confirmed expression of the kanR message and protein in treponemes propagated in vitro. Moreover, tprA knockout (tprAko-SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin. Conclusion We demonstrated that genetic manipulation of T. pallidum is attainable. This discovery will allow the application of functional genomics to study syphilis pathogenesis and improve syphilis vaccine development.

Background An effective syphilis vaccine will be integral to efforts to eradicate this disease. Promising vaccine candidates are surface antigens of the syphilis spirochete, Treponema pallidum subsp. pallidum (T. pallidum). These antigens can be targeted by vaccination-induced opsonic antibodies and mediate pathogen immune clearance. Defining correlates of protection will aid in the identification of the best vaccine candidates. Here, we sought to investigate whether immunization with variants of the T. pallidum Repeat C (TprC) protein and the conserved NH2-terminus of the TprK induced protection and whether splenocyte proliferation and IFN-γ production correlated with protection. Methods Rabbits were immunized with either a cocktail of three recombinant, full-length TprC variants, or the NH2-terminus of the TprK protein with a RIBI-like adjuvant. Animals were challenged with T. pallidum intradermally (10 sites; 10^5 bacteria/site). Treponemal burden and progression to ulceration were monitored. To assess for immunogen-specific splenocytes, pools of synthetic peptides corresponding to each immunogen were used to stimulate splenocytes collected ex-vivo in proliferation assays. Supernatants from stimulated splenocytes were used to quantify IFN-γ responses by ELISA. Results Immunizations protected animals significantly albeit not completely. At day 35 post-challenge only 14.1% and 15.5% of lesions ulcerated in immunized rabbits compared to the 95% of lesions in unimmunized rabbits. At day 21, there was a 99.3% and 98.7% reduction in treponemal burden averaged across all challenge sites in TprC- and TprK-immunized rabbits compared to unimmunized animals, respectively. Lymphocyte proliferation and IFN-γ production correlated to reduction in both percent of ulcerated lesions and treponemal burden. Conclusions Lymphocyte proliferation and IFN-γ release assays may serve as surrogates to assess for antigen-specific T-cell responses. TprK and TprC immunizations are able to stimulate cellular immunity in a TH2 environment, which is key to development of an effective syphilis vaccine.

BackgroundDespite more than a century of research, genetic manipulation ofTreponema pallidumsubsp.pallidum(T. pallidum), the causative agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding of the mechanisms behindT. pallidumsuccess as a pathogen. A recently described method forin vitrocultivation ofT. pallidum,however, has made it possible to experiment with transformation and selection protocols in this pathogen. Here, we describe an approach that successfully replaced thetprA(tp0009) pseudogene in the SS14T. pallidumstrain with a kanamycin resistance (kanR) cassette.Principal findingsA suicide vector was constructed using the pUC57 plasmid backbone. In the vector, thekanRgene was cloned downstream of thetp0574gene promoter. Thetp0574prom-kanRcassette was then placed between two 1-kbp homology arms identical to the sequences upstream and downstream of thetprApseudogene. To induce homologous recombination and integration of thekanRcassette into theT. pallidumchromosome,in vitro-cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl2-based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of thekanRcassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole-genome sequencing (WGS) of transformed treponemes propagatedin vitroandin vivo. ddPCR analysis of RNA and mass spectrometry confirmed expression of thekanRmessage and protein in treponemes propagatedin vitro. Moreover,tprAknockout (tprAko-SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin.ConclusionWe demonstrated that genetic manipulation ofT. pallidumis attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development.Author SummarySyphilis is still an endemic disease in many low- and middle-income countries, and it has been resurgent in high-income nations for almost two decades. In endemic areas, syphilis causes significant morbidity and mortality, particularly when its causative agent, the spirocheteTreponema pallidumsubsp. pallidum(T. pallidum) is transmitted to the fetus during pregnancy. A better understanding ofT. pallidumbiology and syphilis pathogenesis would help devise better control strategies for this infection. One of the limitations associated with working withT. pallidumwas our inability to genetically alter this pathogen to evaluate the function of genes encoding virulence factors or create attenuated strains that could be useful for vaccine development. Here, we report a transformation protocol that allowed us to replace a specific region of theT. pallidumgenome containing a pseudogene (i.e., a non-functional gene) with a stably integrated kanamycin resistance gene. To our knowledge, this is the first-ever report of a method to achieve a genetically modifiedT. pallidumstrain and, as such, it can revolutionize research in the syphilis field.

BackgroundAn effective syphilis vaccine should elicit antibodies to Treponema pallidum subsp. pallidum (T. p. pallidum) surface antigens to induce pathogen clearance through opsonophagocytosis. Although the combination of bioinformatics, structural, and functional analyses of T. p. pallidum genes to identify putative outer membrane proteins (OMPs) resulted in a list of potential vaccine candidates, still very little is known about whether and how transcription of these genes is regulated during infection. This knowledge gap is a limitation to vaccine design, as immunity generated to an antigen that can be down-regulated or even silenced at the transcriptional level without affecting virulence would not induce clearance of the pathogen, hence allowing disease progression.Principal findingsWe report here that tp1031, the T. p. pallidum gene encoding the putative OMP and vaccine candidate TprL is differentially expressed in several T. p. pallidum strains, suggesting transcriptional regulation. Experimental identification of the tprL transcriptional start site revealed that a homopolymeric G sequence of varying length resides within the tprL promoter and that its length affects promoter activity compatible with phase variation. Conversely, in the closely related pathogen T. p. subsp. pertenue, the agent of yaws, where a naturally-occurring deletion has eliminated the tprL promoter region, elements necessary for protein synthesis, and part of the gene ORF, tprL transcription level are negligible compared to T. p. pallidum strains. Accordingly, the humoral response to TprL is absent in yaws-infected laboratory animals and patients compared to syphilis-infected subjects.ConclusionThe ability of T. p. pallidum to stochastically vary tprL expression should be considered in any vaccine development effort that includes this antigen. The role of phase variation in contributing to T. p. pallidum antigenic diversity should be further studied.Author SummarySyphilis is still an endemic disease in many low- and middle-income countries and has been resurgent in high-income nations for almost two decades now. In endemic areas, syphilis still causes significant morbidity and mortality in patients, particularly when its causative agent, the bacterium Treponema pallidum subsp. pallidum is transmitted to the fetus during pregnancy. Although there are significant ongoing efforts to identify an effective syphilis vaccine to bring into clinical trials within the decade in the U.S., such efforts are partially hindered by the lack of knowledge on transcriptional regulation of many genes encoding vaccine candidates. Here, we start addressing this knowledge gap for the putative outer membrane protein (OMP) and vaccine candidates TprL, encoded by the tp1031 gene. As we previously reported for other putative OMP-encoding genes of the syphilis agent, tprL transcription level appears to be affected by the length of a homopolymeric sequence of guanosines (Gs) located within the gene promoter. This is a mechanism known as phase variation and often involved in altering the surface antigenic profile of a bacterial pathogen to facilitate immune evasion and/or adaptation to the host milieu.