Genetic engineering of Treponema pallidum subsp. pallidum, the Syphilis Spirochete.
Journal
PLoS pathogens
ISSN: 1553-7374
Titre abrégé: PLoS Pathog
Pays: United States
ID NLM: 101238921
Informations de publication
Date de publication:
07 2021
07 2021
Historique:
received:
02
05
2021
accepted:
21
06
2021
revised:
16
07
2021
pubmed:
7
7
2021
medline:
25
2
2023
entrez:
6
7
2021
Statut:
epublish
Résumé
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 (kanR) cassette. 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 and integration of the kanR cassette into the T. pallidum chromosome, in vitro-cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl2-based 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 transformed treponemes propagated in vitro and/or 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. 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.
Identifiants
pubmed: 34228757
doi: 10.1371/journal.ppat.1009612
pii: PPATHOGENS-D-21-00942
pmc: PMC8284648
doi:
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
e1009612Subventions
Organisme : NIAID NIH HHS
ID : U19 AI144133
Pays : United States
Déclaration de conflit d'intérêts
The authors have declared that no competing interests exist.
Références
Sex Transm Dis. 2005 Apr;32(4):220-6
pubmed: 15788919
BMC Struct Biol. 2018 May 16;18(1):7
pubmed: 29769048
Front Microbiol. 2018 Nov 26;9:2819
pubmed: 30534115
Science. 1998 Jul 17;281(5375):375-88
pubmed: 9665876
Nat Rev Mol Cell Biol. 2013 Jan;14(1):49-55
pubmed: 23169466
Curr Protoc Microbiol. 2007 Nov;Chapter 12:Unit 12A.1
pubmed: 18770607
Bioinformatics. 2012 Jun 15;28(12):1647-9
pubmed: 22543367
Infect Immun. 2007 Jan;75(1):104-12
pubmed: 17030565
Infect Immun. 1981 May;32(2):908-15
pubmed: 7019081
Genome Announc. 2014 Apr 17;2(2):
pubmed: 24744342
Microbiol Mol Biol Rev. 2016 Mar 30;80(2):411-27
pubmed: 27030552
Curr Protoc Microbiol. 2005 Jul;Chapter 12:Unit 12B.2
pubmed: 18770552
Nat Rev Genet. 2014 May;15(5):321-34
pubmed: 24690881
Bioinformatics. 2014 Aug 1;30(15):2114-20
pubmed: 24695404
Am J Obstet Gynecol. 2003 Sep;189(3):861-73
pubmed: 14526331
Mol Biol Evol. 2006 Nov;23(11):2220-33
pubmed: 16926243
J Bacteriol. 2008 Apr;190(7):2565-71
pubmed: 18263731
J Bacteriol. 2002 Jun;184(12):3194-202
pubmed: 12029035
Sci Rep. 2016 May 10;6:25593
pubmed: 27161310
Curr Opin Infect Dis. 2011 Feb;24(1):50-5
pubmed: 21150594
J Bacteriol. 2005 Mar;187(5):1866-74
pubmed: 15716460
Oral Microbiol Immunol. 1996 Jun;11(3):161-5
pubmed: 8941770
Curr Protoc. 2021 Feb;1(2):e44
pubmed: 33599121
J Vet Sci. 2006 Jun;7(2):111-7
pubmed: 16645333
Front Microbiol. 2019 Jul 31;10:1691
pubmed: 31417509
J Immunol. 2010 Apr 1;184(7):3822-9
pubmed: 20190145
Infect Genet Evol. 2012 Mar;12(2):191-202
pubmed: 22198325
PLoS Negl Trop Dis. 2014 Nov 06;8(11):e3261
pubmed: 25375929
Annu Rev Genet. 1989;23:37-69
pubmed: 2694936
Nat Methods. 2012 Mar 04;9(4):357-9
pubmed: 22388286
J Immunol. 1988 Dec 15;141(12):4363-9
pubmed: 2461990
Sex Transm Dis. 1988 Jan-Mar;15(1):70
pubmed: 3358241
Annu Rev Genet. 2011;45:273-97
pubmed: 22060043
Euro Surveill. 2009 Nov 26;14(47):
pubmed: 19941803
J Bacteriol. 2005 Sep;187(17):6084-93
pubmed: 16109950
mBio. 2021 Feb 23;12(1):
pubmed: 33622721
PLoS Pathog. 2011 Sep;7(9):e1002258
pubmed: 21966270
Euro Surveill. 2012 Jul 19;17(29):
pubmed: 22835469
Nat Microbiol. 2016 Dec 05;2:16245
pubmed: 27918528
Lancet. 1998;351 Suppl 3:2-4
pubmed: 9652711
Infect Immun. 1992 Apr;60(4):1568-76
pubmed: 1372297
Clin Microbiol Rev. 2006 Jan;19(1):29-49
pubmed: 16418521
Infect Immun. 1999 Jul;67(7):3653-6
pubmed: 10377154
Rev Infect Dis. 1988 Jul-Aug;10 Suppl 2:S274-6
pubmed: 3055197
J Bacteriol. 2020 Mar 11;202(7):
pubmed: 31932313
Annu Rev Biochem. 2013;82:25-54
pubmed: 23746253
Med J Aust. 2005 Aug 15;183(4):179-83
pubmed: 16097913
Cold Spring Harb Perspect Biol. 2013 Oct 01;5(10):a010116
pubmed: 23838439
mBio. 2018 Jun 26;9(3):
pubmed: 29946052
Cold Spring Harb Perspect Biol. 2013 Apr 01;5(4):a012922
pubmed: 23471435