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

Authors :
Romeis, Emily
Tantalo, Lauren
Lieberman, Nicole
Phung, Quynh
Greninger, Alex
Giacani, Lorenzo
Source :
PLoS Pathogens; 7/6/2021, Vol. 17 Issue 7, p1-25, 25p
Publication Year :
2021

Abstract

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<superscript>R</superscript>) cassette. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kan<superscript>R</superscript> gene was cloned downstream of the tp0574 gene promoter. The tp0574prom-kan<superscript>R</superscript> 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<superscript>R</superscript> cassette into the T. pallidum chromosome, in vitro-cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl<subscript>2</subscript>-based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kan<superscript>R</superscript> 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<superscript>R</superscript> message and protein in treponemes propagated in vitro. Moreover, tprA knockout (tprA<superscript>ko</superscript>-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. Author summary: Syphilis 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 spirochete Treponema pallidum subsp. pallidum (T. pallidum) is transmitted to the fetus during pregnancy. A better understanding of T. pallidum biology and syphilis pathogenesis would help devise better control strategies for this infection. One of the limitations associated with working with T. pallidum was our inability to genetically alter this pathogen to evaluate the function of genes encoding virulence factors or create attenuated strains that could be informative for vaccine development when studied using the rabbit model of the disease. Here, we report a transformation protocol that allowed us to replace a specific region of the T. pallidum genome 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 modified T. pallidum strain. [ABSTRACT FROM AUTHOR]

Details

Language :
English
ISSN :
15537366
Volume :
17
Issue :
7
Database :
Complementary Index
Journal :
PLoS Pathogens
Publication Type :
Academic Journal
Accession number :
151269984
Full Text :
https://doi.org/10.1371/journal.ppat.1009612