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Updated: Jul 9, 2025
Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking
Published on: December 17, 2013
Lysinoalanine cross-linking is a conserved post-translational modification in the spirochete flagellar hook
Michael J Lynch1, Maithili Deshpande1, Kurni Kurniyati2
1Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA.
Abstract:
Spirochetes cause Lyme disease, leptospirosis, syphilis, and several other human illnesses. Unlike other bacteria, spirochete flagella are enclosed within the periplasmic space where the filaments distort and push the cell body by the action of the flagellar motors. We previously demonstrated that the oral pathogen Treponema denticola (Td) and Lyme disease pathogen Borreliella burgdorferi (Bb) form covalent lysinoalanine (Lal) cross-links between conserved cysteine and lysine residues of the FlgE protein that composes the flagellar hook. In Td, Lal is unnecessary for hook assembly but is required for motility, presumably due to the stabilizing effect of the cross-link. Herein, we extend these findings to other, representative spirochete species across the phylum. We confirm the presence of Lal cross-linked peptides in recombinant and in vivo-derived samples from Treponema spp., Borreliella spp., Brachyspira spp., and Leptospira spp. As was observed with Td, a mutant strain of Bb unable to form the cross-link has greatly impaired motility. FlgE from Leptospira spp. does not conserve the Lal-forming cysteine residue which is instead substituted by serine. Nevertheless, Leptospira interrogans FlgE also forms Lal, with several different Lal isoforms being detected between Ser-179 and Lys-145, Lys-148, and Lys-166, thereby highlighting species or order-specific differences within the phylum. Our data reveal that the Lal cross-link is a conserved and necessary posttranslational modification across the spirochete phylum and may thus represent an effective target for the development of spirochete-specific antimicrobials.
Insights
Lysinoalanine (Lal) cross-links are essential for spirochete motility across diverse species. This conserved modification in flagellar proteins offers a potential target for novel antimicrobial therapies against diseases like Lyme disease and leptospirosis.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Spirochetes are pathogenic bacteria responsible for diseases such as Lyme disease, leptospirosis, and syphilis.
- Unlike other bacteria, spirochete flagella are located within the periplasmic space, enabling motility through unique mechanisms.
- Previous research identified covalent lysinoalanine (Lal) cross-links in the flagellar hook protein (FlgE) of *Treponema denticola* and *Borreliella burgdorferi*.
Purpose of the Study:
- To investigate the presence and necessity of lysinoalanine (Lal) cross-links in the flagellar hook protein (FlgE) across various spirochete species.
- To determine if Lal cross-links are a conserved posttranslational modification within the Spirochete phylum.
- To evaluate the potential of Lal cross-links as a target for developing spirochete-specific antimicrobials.
Main Methods:
- Analysis of recombinant and in vivo-derived samples from multiple spirochete species (*Treponema*, *Borreliella*, *Brachyspira*, *Leptospira*).
- Mass spectrometry to identify and characterize lysinoalanine (Lal) cross-linked peptides in FlgE.
- Motility assays using wild-type and mutant spirochete strains lacking the ability to form Lal cross-links.
Main Results:
- Lysinoalanine (Lal) cross-linked peptides were confirmed in FlgE from *Treponema* spp., *Borreliella* spp., *Brachyspira* spp., and *Leptospira* spp.
- A *Borreliella burgdorferi* mutant unable to form Lal cross-links exhibited significantly impaired motility.
- While *Leptospira* spp. FlgE lacks the conserved cysteine residue for Lal formation, alternative Lal isoforms were detected, indicating species-specific variations.
Conclusions:
- The lysinoalanine (Lal) cross-link is a conserved and essential posttranslational modification for flagellar function and motility across the Spirochete phylum.
- The presence of Lal cross-links in diverse spirochetes suggests they are crucial for the structural integrity and function of the flagellar hook.
- Lal cross-links represent a promising and conserved target for the development of novel, spirochete-specific antimicrobial agents.
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