Advertisement

Genetic Manipulations of Oral Spirochete Treponema denticola

Protocol
  • 532 Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 2210)

Abstract

There have been more than 60 different oral Treponema species identified in the oral cavity; however, only few species can be cultivated in vitro reliably. Among those cultivable species, due to its medical importance and genetic tractability, Treponema denticola, one of the keystone pathogens associated with human periodontitis, has emerged as a paradigm model organism to understanding the genetics, etiology, and pathophysiology of oral Treponema species. During the last two decades, several genetic tools have been developed, which have played an instrumental role in the study of T. denticola. This chapter describes the experimental design and procedure of genetic manipulations of T. denticola.

Key words

Periodontal disease Spirochete Treponema denticola Genetic manipulation 

References

  1. 1.
    Ellen RP, Galimanas VB (2005) Spirochetes at the forefront of periodontal infections. Periodontol 2000 38:13–32CrossRefGoogle Scholar
  2. 2.
    Holt SC, Ebersole JL (2005) Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia: the “red complex”, a prototype polybacterial pathogenic consortium in periodontitis. Periodontol 2000 38:72–122CrossRefGoogle Scholar
  3. 3.
    Darveau RP (2010) Periodontitis: a polymicrobial disruption of host homeostasis. Nat Rev Microbiol 8:481–490CrossRefGoogle Scholar
  4. 4.
    Dashper SG, Seers CA, Tan KH et al (2011) Virulence factors of the oral spirochete Treponema denticola. J Dent Res 90:691–703CrossRefGoogle Scholar
  5. 5.
    Fenno JC, McBride BC (1998) Virulence factors of oral treponemes. Anaerobe 4:1–17CrossRefGoogle Scholar
  6. 6.
    Li H, Ruby J, Charon N et al (1996) Gene inactivation in the oral spirochete Treponema denticola: construction of an flgE mutant. J Bacteriol 178:3664–3667CrossRefGoogle Scholar
  7. 7.
    Li H, Kuramitsu HK (1996) Development of a gene transfer system in Treponema denticola by electroporation. Oral Microbiol Immunol 11:161–165CrossRefGoogle Scholar
  8. 8.
    Yang Y, Stewart PE, Shi X et al (2008) Development of a transposon mutagenesis system in the oral spirochete Treponema denticola. Appl Environ Microbiol 74:6461–6464CrossRefGoogle Scholar
  9. 9.
    Li Y, Ruby J, Wu H (2015) Kanamycin resistance cassette for the genetic manipulation of Treponema denticola. Appl Environ Microbiol 13:4329–4338CrossRefGoogle Scholar
  10. 10.
    Goetting-Minesky MP, Fenno JC (2010) A simplified erythromycin resistance cassette for Treponema denticola mutagenesis. J Microbiol Methods 83:66–68CrossRefGoogle Scholar
  11. 11.
    Chi B, Limberger RJ, Kuramitsu HK (2002) Complementation of a Treponema denticola flgE mutant with a novel coumermycin A1-resistant T. denticola shuttle vector system. Infect Immun 70:2233–2237CrossRefGoogle Scholar
  12. 12.
    Chi B, Chauhan S, Kuramitsu H (1999) Development of a system for expressing heterologous genes in the oral spirochete Treponema denticola and its use in expression of the Treponema pallidum flaA gene. Infect Immun 67:3653–3656CrossRefGoogle Scholar
  13. 13.
    Bian J, Fenno JC, Li C (2012) Development of a modified gentamicin resistance cassette for genetic manipulation of the oral spirochete Treponema denticola. Appl Environ Microbiol 78:2059–2062CrossRefGoogle Scholar
  14. 14.
    Kurniyati K, Li C (2016) pyrF as a counterselectable marker for unmarked genetic manipulations in Treponema denticola. Appl Environ Microbiol 82:1346–1352CrossRefGoogle Scholar
  15. 15.
    Slivienski-Gebhardt LL, Izard J, Samsonoff WA et al (2004) Development of a novel chloramphenicol resistance expression plasmid used for genetic complementation of a fliG deletion mutant in Treponema denticola. Infect Immun 72:5493–5497CrossRefGoogle Scholar
  16. 16.
    Asif A, Mohsin H, Tanvir R et al (2017) Revisiting the mechanisms involved in calcium chloride induced bacterial transformation. Front Microbiol 8:2169CrossRefGoogle Scholar
  17. 17.
    Ohta K, Makinen KK, Loesche WJ (1986) Purification and characterization of an enzyme produced by Treponema denticola capable of hydrolyzing synthetic trypsin substrates. Infect Immun 53:213–220CrossRefGoogle Scholar
  18. 18.
    Seshadri R, Myers GS, Tettelin H et al (2004) Comparison of the genome of the oral pathogen Treponema denticola with other spirochete genomes. Proc Natl Acad Sci U S A 101:5646–5651CrossRefGoogle Scholar
  19. 19.
    Orth R, O’Brien-Simpson N, Dashper S et al (2010) An efficient method for enumerating oral spirochetes using flow cytometry. J Microbiol Methods 80:123–128CrossRefGoogle Scholar
  20. 20.
    Cha-Aim K, Hoshida H, Fukunaga T et al (2012) Fusion PCR via novel overlap sequences. Methods Mol Biol 852:97–110CrossRefGoogle Scholar
  21. 21.
    Godovikova V, Goetting-Minesky MP, Shin JM et al (2015) A modified shuttle plasmid facilitates expression of a flavin mononucleotide-based fluorescent protein in Treponema denticola ATCC 35405. Appl Environ Microbiol 81:6496–6504CrossRefGoogle Scholar
  22. 22.
    Bian J, Li C (2011) Disruption of a type II endonuclease (TDE0911) enables Treponema denticola ATCC 35405 to accept an unmethylated shuttle vector. Appl Environ Microbiol 77:4573–4578CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  1. 1.Department of Oral and Craniofacial Molecular Biology, Philips Institute for Oral Health ResearchVirginia Commonwealth UniversityRichmondUSA

Personalised recommendations