O01.7 At Long Last: Genetic Engineering of Treponema pallidum subsp. pallidum, the Syphilis Spirochete

Abstract

<h3>Background</h3> 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. <h3>Principal findings</h3> 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. <h3>Conclusion</h3> 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.