Despite 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 (kan^R) cassette. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, thekan^Rgene was cloned downstream of thetp0574gene promoter. Thetp0574prom-kan^Rcassette 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 thekan^Rcassette 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 thekan^Rcassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole-genome sequencing (WGS) of transformed treponemes propagatedin vitroand/orin vivo. ddPCR analysis of RNA and mass spectrometry confirmed expression of thekan^Rmessage and protein in treponemes propagatedin vitro. Moreover,tprAknockout (tprA^ko-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 ofT.pallidumis attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development.

Although the threeTreponema pallidumsubspecies (T. pallidumsubsp.pallidum,T. pallidumsubsp.pertenue, andT. pallidumsubsp.endemicum),Treponema paraluiscuniculi, and the unclassified Fribourg-Blanc treponeme cause clinically distinct diseases, these pathogens are genetically and antigenically highly related and are able to cause persistent infection. Recent evidence suggests that the putative surface-exposed variable antigen TprK plays an important role in both treponemal immune evasion and persistence.tprKheterogeneity is generated by nonreciprocal gene conversion between thetprKexpression site and donor sites. Although each of the above-mentioned species and subspecies has a functionaltprKantigenic variation system, it is still unclear why the level of expression and the rate at whichtprKdiversifies during infection can differ significantly among isolates. To identify genomic differences that might affect the generation and expression of TprK variants among these pathogens, we performed comparative sequence analysis of the donor sites, as well as thetprKexpression sites, among eightT. pallidumsubsp.pallidumisolates (Nichols Gen, Nichols Sea, Chicago, Sea81-4, Dal-1, Street14, UW104, and UW126), threeT. pallidumsubsp.pertenueisolates (Gauthier, CDC2, and Samoa D), oneT. pallidumsubsp.endemicumisolate (Iraq B), the unclassified Fribourg-Blanc isolate, and the Cuniculi A strain ofT. paraluiscuniculi. Synteny and sequence conservation, as well as deletions and insertions, were found in the regions harboring the donor sites. These data suggest that thetprKrecombination system is harbored within dynamic genomic regions and that genomic differences might be an important key to explain discrepancies in generation and expression oftprKvariants among theseTreponemaisolates.

The increasing incidence of syphilis and the limitations of first-line treatment with penicillin, particularly in neurosyphilis, neonatal syphilis, and pregnancy, highlight the need to expand the therapeutic repertoire for effective management of this disease. We assessed the in-vitro efficacy of 18 antibiotics from several classes on Treponema pallidum subspecies pallidum (T pallidum), the syphilis bacteria. Using the in-vitro culture system for T pallidum, we exposed the pathogen to a concentration range of each tested antibiotic. After a 7-day incubation, the treponemal burden was evaluated by quantitative PCR targeting the T pallidum tp0574 gene. The primary outcome was the minimum inhibitory concentration (MIC) at which the quantitative PCR values were not significantly higher than the inoculum wells. We also investigated the susceptibility of macrolide-resistant strains to high concentrations of azithromycin, and the possibility of developing resistance to linezolid, a proposed candidate for syphilis treatment. Amoxicillin, ceftriaxone, several oral cephalosporins, tedizolid, and dalbavancin exhibited anti-treponemal activity at concentrations achievable in human plasma following regular dosing regimens. The experiments revealed a MIC for amoxicillin at 0·02 mg/L, ceftriaxone at 0·0025 mg/L, cephalexin at 0·25 mg/L, cefetamet and cefixime at 0·0313 mg/L, cefuroxime at 0·0156 mg/L, tedizolid at 0·0625 mg/L, spectinomycin at 0·1 mg/L, and dalbavancin at 0·125 mg/L. The MIC for zoliflodacin and balofloxacin was 2 mg/L. Ertapenem, isoniazid, pyrazinamide, and metronidazole had either a poor or no effect. Azithromycin concentrations up to 2 mg/L (64 times the MIC) were ineffective against strains carrying mutations associated to macrolide resistance. Exposure to subtherapeutic doses of linezolid for 10 weeks did not induce phenotypic or genotypic resistance. Cephalosporins and oxazolidinones are potential candidates for expanding the current therapeutic repertoire for syphilis. Our findings warrant testing efficacy in animal models and, if successful, clinical assessment of efficacy. European Research Council.

In syphilis research, the Nichols strain of Treponema pallidum , isolated in 1912, has been the most widely studied. Recently, important differences among T. pallidum strains emerged; therefore, we sequenced and annotated the Chicago strain genome to facilitate and encourage the use of this strain in studying the pathogenesis of syphilis.

Penicillin G, the current standard treatment for syphilis, has important drawbacks, but virtually no preclinical or clinical studies have been performed to identify viable alternatives. We tested, both in vitro and in vivo, three marketed antibiotics with adequate pharmacological properties to treat syphilis. We used an in vitro culturing system of T. pallidum to perform drug susceptibility testing and applied quantitative PCR targeting the tp0574 gene to measure bacterial growth. To confirm in vivo efficacy, fifteen rabbits were infected intradermally with T. pallidum at eight sites each and randomly allocated to an experimental treatment (linezolid, moxifloxacin, clofazimine) or a control arm (benzathine penicillin G [BPG], untreated). The primary outcome was treatment efficacy defined as the time to lesion healing measured from the date of treatment start. Secondary outcomes were absence of treponemes or treponemal mRNA in injection sites, absence of seroconversion, and cerebrospinal fluid (CSF) abnormalities and negative rabbit infectivity tests (RIT). Linezolid showed in vitro bactericidal activity at concentrations of 0.5 µg/mL or higher. When administered orally to experimentally infected rabbits, it induced healing of early lesions at a time similar to BPG (hazard ratio 3.84; 95% CI 2.05-7.17; p < 0.0001 compared to untreated controls). In linezolid-treated animals, dark-field microscopy and qPCR assessment showed no presence of treponemes after day 3 post-treatment start, serologic test did not convert to positive, CSF had no abnormalities, and RIT was negative. Moxifloxacin and clofazimine failed to inhibit bacterial growth in vitro and could not cure the infection in the rabbit model. Linezolid, a low-cost oxazolidinone, has in vitro and in vivo activity against T. pallidum, with efficacy similar to BPG in treating treponemal lesions in the animal model. Our findings warrant further research to assess the efficacy of linezolid as an alternative to penicillin G to treat syphilis in human clinical trials. European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (Grant agreement No. 850450).