Letter | Published:

Spirochaete flagella hook proteins self-catalyse a lysinoalanine covalent crosslink for motility

Nature Microbiology volume 1, Article number: 16134 (2016) | Download Citation

Abstract

Spirochaetes are bacteria responsible for several serious diseases, including Lyme disease (Borrelia burgdorferi), syphilis (Treponema pallidum) and leptospirosis (Leptospira interrogans), and contribute to periodontal diseases (Treponema denticola)1. These spirochaetes employ an unusual form of flagella-based motility necessary for pathogenicity; indeed, spirochaete flagella (periplasmic flagella) reside and rotate within the periplasmic space2,​3,​4,​5,​6,​7,​8,​9,​10,​11. The universal joint or hook that links the rotary motor to the filament is composed of 120–130 FlgE proteins, which in spirochaetes form an unusually stable, high-molecular-weight complex9,12,​13,​14,​15,​16,​17. In other bacteria, the hook can be readily dissociated by treatments such as heat18. In contrast, spirochaete hooks are resistant to these treatments, and several lines of evidence indicate that the high-molecular-weight complex is the consequence of covalent crosslinking12,13,17. Here, we show that T. denticola FlgE self-catalyses an interpeptide crosslinking reaction between conserved lysine and cysteine, resulting in the formation of an unusual lysinoalanine adduct that polymerizes the hook subunits. Lysinoalanine crosslinks are not needed for flagellar assembly, but they are required for cell motility and hence infection. The self-catalytic nature of FlgE crosslinking has important implications for protein engineering, and its sensitivity to chemical inhibitors provides a new avenue for the development of antimicrobials targeting spirochaetes.

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References

  1. 1.

    in Bergey's Manuel of Systematic Bacteriology Vol. 4 (eds Krieg, N. R. et al.), 471–566 (Springer, 2011).

  2. 2.

    et al. Motility is crucial for the infectious life cycle of Borrelia burgdorferi. Infect. Immun. 81, 2012–2021 (2013).

  3. 3.

    , , & Inactivation of a putative flagellar motor switch protein FliG1 prevents Borrelia burgdorferi from swimming in highly viscous media and blocks its infectivity. Mol. Microbiol. 75, 1563–1576 (2010).

  4. 4.

    , , , & Borrelia burgdorferi needs chemotaxis to establish infection in mammals and to accomplish its enzootic cycle. Infect. Immun. 80, 2485–2492 (2012).

  5. 5.

    et al. FlaA proteins in Leptospira interrogans are essential for motility and virulence but are not required for formation of the flagellum sheath. Infect. Immun. 80, 2019–2025 (2012).

  6. 6.

    et al. Inactivation of the fliY gene encoding a flagellar motor switch protein attenuates mobility and virulence of Leptospira interrogans strain Lai. BMC Microbiol. 9, 253 (2009).

  7. 7.

    et al. Motor rotation is essential for the formation of the periplasmic flagellar ribbon, cellular morphology, and Borrelia burgdorferi persistence within Ixodes scapularis tick and murine hosts. Infect. Immun. 83, 1765–1777 (2015).

  8. 8.

    & Genetics of motility and chemotaxis of a fascinating group of bacteria: the spirochetes. Annu. Rev. Genet. 36, 47–73 (2002).

  9. 9.

    et al. The unique paradigm of spirochete motility. Annu. Rev. Microbiol. 66, 349–370 (2012).

  10. 10.

    Flagellar motility of the pathogenic spirochetes. Semin. Cell Dev. Biol. 46, 104–112 (2015).

  11. 11.

    et al. A novel flagellar sheath protein, FcpA, determines filament coiling, translational motility and virulence for the Leptospira spirochete. Mol. Microbiol. (24 May 2016).

  12. 12.

    et al. Initial characterization of the FlgE hook high molecular weight complex of Borrelia burgdorferi. PLoS ONE 9, e98338 (2014).

  13. 13.

    , & Genetic and biochemical analysis of the flagellar hook of Treponema phagedenis. J. Bacteriol. 176, 3631–3637 (1994).

  14. 14.

    , , & Insertional inactivation of Treponema denticola tap1 results in a nonmotile mutant with elongated flagellar hooks. J. Bacteriol. 181, 3743–3750 (1999).

  15. 15.

    , & Complementation of a Treponema denticola flgE mutant with a novel coumermycin A1-resistant T. denticola shuttle vector system. Infect. Immun. 70, 2233–2237 (2002).

  16. 16.

    , , & Organization, transcription, and expression of the 5' region of the fla operon of Treponema phagedenis and Treponema pallidum. J. Bacteriol. 178, 4628–4634 (1996).

  17. 17.

    et al. Borrelia burgdorferi uniquely regulates its motility genes and has an intricate flagellar hook-basal body structure. J. Bacteriol. 190, 1912–1921 (2008).

  18. 18.

    , , & Stoichiometric analysis of the flagellar hook-(basal-body) complex of Salmonella typhimurium. J. Mol. Biol. 212, 377–387 (1990).

  19. 19.

    , , & Bacteriophage HK97 structure: wholesale covalent cross-linking between the major head shell subunits. J. Virol. 65, 3227–3237 (1991).

  20. 20.

    Characterization of the Unique Flagellar Hook Structure of the Spirochetes Borrelia burgdorferi and Treponema denticola PhD thesis (West Virginia Univ., 2013).

  21. 21.

    Chemistry, biochemistry, nutrition, and microbiology of lysinoalanine, lanthionine, and histidinoalanine in food and other proteins. J. Agric. Food Chem. 47, 1295–1319 (1999).

  22. 22.

    , & Specific arrangement of alpha-helical coiled coils in the core domain of the bacterial flagellar hook for the universal joint function. Structure 17, 1485–1493 (2009).

  23. 23.

    et al. Relationship of Treponema denticola periplasmic flagella to irregular cell morphology. J. Bacteriol. 179, 1628–1635 (1997).

  24. 24.

    , , & Gene inactivation in the oral spirochete Treponema denticola: construction of a flgE mutant. J. Bacteriol. 178, 3664–3667 (1996).

  25. 25.

    , & Bacteria can exploit a flagellar buckling instability to change direction. Nature Phys. 9, 494–498 (2013).

  26. 26.

    et al. Structure and energetics of encapsidated DNA in bacteriophage HK97 studied by scanning calorimetry and cryo-electron microscopy. J. Mol. Biol. 391, 471–483 (2009).

  27. 27.

    , & Self-generated covalent cross-links in the cell-surface adhesins of Gram-positive bacteria. Biochem. Soc. Trans. 43, 787–794 (2015).

  28. 28.

    & Localization of the transglutaminase cross-linking sites in the Bacillus subtilis spore coat protein GerQ. J. Bacteriol. 188, 7609–7616 (2006).

  29. 29.

    , , & Nine post-translational modifications during the biosynthesis of cinnamycin. J. Am. Chem. Soc. 133, 13753–13760 (2011).

  30. 30.

    , , , & An efficient method for enumerating oral spirochetes using flow cytometry. J. Microbiol. Meth. 80, 123–128 (2010).

  31. 31.

    , , , & The riboswitch regulates a thiamine pyrophosphate ABC transporter of the oral spirochete Treponema denticola. J. Bacteriol. 193, 3912–3922 (2011).

  32. 32.

    et al. Proton NMR measurements of bacteriophage T4 lysozyme aided by 15N isotopic labeling: structural and dynamic studies of larger proteins. Proc. Natl Acad. Sci. USA 84, 1244–1248 (1987).

  33. 33.

    , , , & Structural organization and assembly of flagellar hook protein from Salmonella typhimurium. J. Mol. Biol. 251, 520–532 (1995).

  34. 34.

    , & Development of a modified gentamicin resistance cassette for genetic manipulation of the oral spirochete Treponema denticola. Appl. Environ. Microbiol. 78, 2059–2062 (2012).

  35. 35.

    & Disruption of a type II endonuclease (TDE0911) enables Treponema denticola ATCC 35405 to accept an unmethylated shuttle vector. Appl. Environ. Microbiol. 77, 4573–4578 (2011).

  36. 36.

    & A simplified erythromycin resistance cassette for Treponema denticola mutagenesis. J. Microbiol. Meth. 83, 66–68 (2010).

  37. 37.

    Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970).

  38. 38.

    & Antiserum to the 33,000-dalton periplasmic-flagellum protein of ‘Treponema phagedenis’ reacts with other treponemes and Spirochaeta aurantia. J. Bacteriol. 168, 1030–1032 (1986).

  39. 39.

    , , & Inactivation of cyclic di-GMP binding protein TDE0214 affects the motility, biofilm formation, and virulence of Treponema denticola. J. Bacteriol. 195, 3897–3905 (2013).

  40. 40.

    Gels, (2015).

  41. 41.

    , , & Development of an integrated approach for evaluation of 2-D gel image analysis: impact of multiple proteins in single spots on comparative proteomics in conventional 2-D gel/MALDI workflow. Electrophoresis 28, 2080–2094 (2007).

  42. 42.

    et al. Evaluation of different multidimensional LC-MS/MS pipelines for isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis of potato tubers in response to cold storage. J. Proteome Res. 10, 4647–4660 (2011).

  43. 43.

    , , , & Monoubiquitination of nuclear RelA negatively regulates NF-κB activity independent of proteasomal degradation. Cell. Mol. Life Sci. 69, 2057–2073 (2012).

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Acknowledgements

Funding was provided by the National Institutes of Health (R01-DE023431, to N.C., M.M. and C.L.), R01 GM064664 (to B.C.), R01-AI087946 (to J.L.) and R01-DE023080 and R01-AI078958 (to C.L). J.L. was also supported by grant AU-1714 from the Welch Foundation. The authors thank B. Bachert, M. Barbier, R. Duda, R. Hendrix, D. McNitt, S. Norris, R. Silversmith and R. Sircar for suggestions, technical assistance and support. The findings and conclusions in this article are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention or the National Institute for Occupational Safety and Health.

Author information

Affiliations

  1. Department of Biochemistry, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, West Virginia 26506, USA

    • Michael R. Miller
  2. Department of Microbiology, Immunology, and Cell Biology, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, West Virginia 26506, USA

    • Kelly A. Miller
    • , Milinda E. James
    • , Andrew Cockburn
    •  & Nyles W. Charon
  3. Department of Oral Biology, State University of New York, Buffalo, New York 14214, USA

    • Jiang Bian
    •  & Chunhao Li
  4. Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, USA

    • Sheng Zhang
    • , Michael J. Lynch
    •  & Brian R. Crane
  5. Department of Pharmaceutical Sciences, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, West Virginia 26506, USA

    • Patrick S. Callery
  6. National Institute for Occupational Safety and Health, 1095 Willowdale Road, Morgantown West Virginia 26505, USA

    • Justin M. Hettick
  7. Department of Pathology and Laboratory Medicine, University of Texas Health Sciences Center, Houston, Texas 77030, USA

    • Jun Liu

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Contributions

N.W.C. and M.R.M. designed the project. B.R.C., N.W.C., C.L, J.L. and M.R.M. wrote the manuscript. P.S.C., N.W.C., B.R.C., J.M.H, M.E.J., C.L., J.L., K.A.M. and M.R.M. designed the experiments. J.B., A.C., J.L., K.A.M., M.R.M., M.E.J., M.L. and S.Z. carried out experiments.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Nyles W. Charon.

Supplementary information

PDF files

  1. 1.

    Supplementary information

    Supplementary Figures 1–12, Supplementary Tables 1–6, Supplementary Video Legends 1–4

Videos

  1. 1.

    Supplementary Video 1

    Cells WT T. denticola in 1% methylcelluose.

  2. 2.

    Supplementary Video 2

    Cells of mutant δflgE in 1% methylcellulose.

  3. 3.

    Supplementary Video 3

    Cells of substitution mutant TdC178A in 1% methylcellulose.

  4. 4.

    Supplementary Video 4

    Cells of substitution mutant TdK169A in 1% methylcellulose.

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DOI

https://doi.org/10.1038/nmicrobiol.2016.134

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