Journal Article

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

Greninger, Alex, Lieberman, Nicole, Phung, Quynh, Tantalo, Lauren, Romeis, Emily, Giacani, Lorenzo

2021

Overview

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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Bacterial infections / Biology and Life Sciences / Calcium chloride / Causes of / Chromosomes / Development and progression / Gene expression / Gene sequencing / Genetic aspects / Genetic engineering / Genetic transformation / Genetically modified organisms / Genetics / Genomes / Homologous recombination / Homology / In vitro methods and tests / Integration / Kanamycin / Mass spectrometry / Mass spectroscopy / Medicine and Health Sciences / Methods / Microbiological research / Minimum inhibitory concentration / Mortality / Pathogenesis / Pathogens / Propagation / Rabbits / Research and Analysis Methods / Selective media / Sexually transmitted diseases / Spirochetes / STD / Suicide / Syphilis / Treponema / Treponema pallidum / Vaccine development / Vaccines / Whole genome sequencing