ArticlePLoS pathogens2021
Genetic engineering of Treponema pallidum subsp. pallidum, the Syphilis Spirochete.
Article in PLoS pathogens, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 43 papers.
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Who cites it
43 citing papers in PubMed, 59 citations in OpenAlex.
- An Adenoviral-Vectored Tp0326 Vaccine Elicits Robust Functional Antibodies to Prevent Treponema pallidum Dissemination in a Rabbit Model.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2026Article
- Putative mechanisms of ocular inflammation in syphilis.Journal of ophthalmic inflammation and infection · 2026Review
- Formal description ofInternational journal of systematic and evolutionary microbiology · 2026Article
- Bacteriophage-Based Therapeutics for Bacterial Sexually Transmitted Infections: From Biological Barriers to Translational Strategies.Pathogens (Basel, Switzerland) · 2026Review
- Lineage-specificbioRxiv : the preprint server for biology · 2026Article
- Expanded Functional Characterization and Optimization of Protein Expression in Treponema denticola Shuttle Plasmids.Molecular oral microbiology · 2026Article
- First human cell-based cultivation system for the syphilis spirochete Treponema pallidum.BMC microbiology · 2026Article
- Review
- A Review of Doxycycline Post-Exposure Prophylaxis and Its Implications for Antimicrobial Resistance and the Human Microbiome.Infectious diseases and therapy · 2026Review
- Sequence variability of BamA and FadL candidate vaccinogens suggests divergent evolutionary paths ofJournal of bacteriology · 2025Article
- Linking genomic evolutionary transitions to ecological phenotypic adaptations inbioRxiv : the preprint server for biology · 2025Article
- High quality transcriptome profiling confirms the transcriptional landscape of Treponema pallidum subsp. pallidum.Scientific reports · 2025Article
- Resistance to ceftriaxone and penicillin G among contemporary syphilis strains confirmed by natural in vitro mutagenesis.Communications medicine · 2025Article
- In vitro isolation of contemporary Treponema pallidum strains directly from clinical samples of syphilis patients.Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases · 2025Article
- Enzyme-constrained metabolic model ofmSystems · 2025Article
- In Vitro Exposure of Treponema pallidum to Subbactericidal Doxycycline Did Not Induce Resistance: Implications for Doxycycline Postexposure Prophylaxis.The Journal of infectious diseases · 2025Article
- The Role of Traditional and Reverse Algorithms in the Diagnosis of Syphilis in HIV-Infected Individuals: A Case Study on Istanbul.Diagnostics (Basel, Switzerland) · 2025Article
- Establishment of the Nichols strain as the type strain ofInternational journal of systematic and evolutionary microbiology · 2025Review
- Development and utilization ofmBio · 2025Article
- Surface nanocoating of bacteria as a versatile platform to develop living therapeutics.Nature protocols · 2024Review
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Authors and funding
6 authors at 1 institution in 1 country.
Funding
Abstract
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.
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