Lysinoalanine crosslinking is a conserved post-translational modification in the spirochete flagellar hook.

Unlabelled: Spirochete bacteria 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 action of the flagellar motors. We previously demonstrated that the oral pathogen (Td) catalyzes the formation of covalent lysinoalanine (Lal) crosslinks between conserved cysteine and lysine residues of the FlgE protein that composes the flagellar hook. Although not necessary for hook assembly, Lal is required for motility of Td, presumably due to the stabilizing effect of the crosslink. Herein, we extend these findings to other, representative spirochete species across the phylum. We confirm the presence of Lal crosslinked peptides in recombinant and -derived samples from spp., spp., spp., and spp.. Like with Td, a mutant strain of the Lyme disease pathogen unable to form the crosslink has impaired motility. FlgE from spp. does not conserve the Lal-forming cysteine residue which is instead substituted by serine. Nevertheless, 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 reveals that the Lal crosslink is a conserved and necessary post-translational modification across the spirochete phylum and may thus represent an effective target for spirochete-specific antimicrobials.

Significance Statement: The phylum Spirochaetota contains bacterial pathogens responsible for a variety of diseases, including Lyme disease, syphilis, periodontal disease, and leptospirosis. Motility of these pathogens is a major virulence factor that contributes to infectivity and host colonization. The oral pathogen produces a post-translational modification (PTM) in the form of a lysinoalanine (Lal) crosslink between neighboring subunits of the flagellar hook protein FlgE. Herein, we demonstrate that representative spirochetes species across the phylum all form Lal in their flagellar hooks. and cells incapable of forming the crosslink are non-motile, thereby establishing the general role of the Lal PTM in the unusual type of flagellar motility evolved by spirochetes.