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

2021; Public Library of Science; Volume: 17; Issue: 7 Linguagem: Inglês

10.1371/journal.ppat.1009612

1553-7374

Emily Romeis, Lauren C. Tantalo, Nicole A. P. Lieberman, Quynh Phung, Alexander L. Greninger, Lorenzo Giacani,

Reproductive tract infections research

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 ( kan R ) cassette. 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 the 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/or 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. 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.