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

Nature Microbiology volume 1, Article number: 16134 (2016) Cite this article

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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 space2–11. 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 complex9,12–17. 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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Figure 1: Stability of FlgE high-molecular-weight complexes (HMWCs) derived from periplasmic flagella (PFs), polyhooks (PHs) and rFlgE.
Figure 2: MS analysis of T. denticola and B. burgdorferi HMWCs indicates that the crosslink is lysinoalanine.
Figure 3: Mutagenesis identifies critical residues for crosslinking that are compatible with structural models of the hook.
Figure 4: T. denticola mutants unable to form FlgE crosslinks are deficient in motility but still form intact flagella.

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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

Authors and Affiliations

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

    Michael R. Miller

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

    Kelly A. Miller, Milinda E. James, Andrew Cockburn & Nyles W. Charon

  3. Department of Oral Biology, State University of New York, Buffalo, 14214, New York, USA

    Jiang Bian & Chunhao Li

  4. Department of Chemistry and Chemical Biology, Cornell University, Ithaca, 14853, New York, USA

    Sheng Zhang, Michael J. Lynch & Brian R. Crane

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

    Patrick S. Callery

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

    Justin M. Hettick

  7. Department of Pathology and Laboratory Medicine, University of Texas Health Sciences Center, Houston, 77030, Texas, 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.

Corresponding author

Correspondence to Nyles W. Charon.

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Supplementary information

Supplementary information

Supplementary Figures 1–12, Supplementary Tables 1–6, Supplementary Video Legends 1–4 (PDF 3951 kb)

Supplementary Video 1

Cells WT T. denticola in 1% methylcelluose. (MOV 5395 kb)

Supplementary Video 2

Cells of mutant δflgE in 1% methylcellulose. (MOV 12170 kb)

Supplementary Video 3

Cells of substitution mutant TdC178A in 1% methylcellulose. (MOV 5836 kb)

Supplementary Video 4

Cells of substitution mutant TdK169A in 1% methylcellulose. (MOV 7137 kb)

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Miller, M., Miller, K., Bian, J. et al. Spirochaete flagella hook proteins self-catalyse a lysinoalanine covalent crosslink for motility. Nat Microbiol 1, 16134 (2016). https://doi.org/10.1038/nmicrobiol.2016.134

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