Genetic Engineering of Treponema pallidum subsp. pallidum, the Syphilis Spirochete

Background.  Despite more than a century of research, genetic manipulation of Treponema pallidum subsp. pallidum ( T. pallidum ), the causative of agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding the mechanisms behind T. pallidum success as a pathogen. A recently described method for in vitro cultivation of T. pallidum, however, has made 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 ( kan R ) cassette.               Principal findings. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kan R gene was cloned downstream of the tp0574 gene promoter. The tp0574 prom- kan R 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 kan R cassette into T. pallidum chromosome, in vitro -cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl 2 -based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kan R cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole genome sequencing (WGS) of transformed treponemes propagated in vitro and in vivo . ddPCR analysis of RNA and mass spectrometry confirmed expression of the kan R message and protein in treponemes propagated in vitro . Moreover, tprA knockout ( 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.             Conclusion. 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.