Genetic Engineering ofTreponema pallidumsubsp.pallidum, the Syphilis Spirochete

AbstractBackgroundDespite 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.