Biology subjects

Charon, N. W.

Publications and source records attributed to Charon, N. W..

3 recordsLinked to original sources

bioRxiv2024

Inhibitors of lysinoalanine crosslinking in the flagella hook as antimicrobials against spirochetes

Spirochetes are especially invasive bacteria that are responsible for several human diseases, including Lyme disease, periodontal disease, syphilis and leptospirosis. Spirochetes rely on an unusual form of motility based on periplasmic flagella (PFs) to infect hosts and evade the immune system. The flexible hook of these PFs contains a post-translational modification in the form of a lysinoalanine (Lal) crosslink between adjacent subunits of FlgE, which primarily comprise the hook. Lal crosslinking has since been found in key species across phylum and involves residues that are highly conserved. The requirement of the Lal crosslink for motility of the pathogens Treponema denticola (Td) and Borreliella burgdorferi (Bb) establish Lal as a potential therapeutic target for the development of anti-microbials. Herein, we present the design, development and application of a NanoLuc-based high-throughput screen that was used to successfully identify two, structurally related Lal crosslink inhibitors (hexachlorophene and triclosan) from a library of clinically approved small molecules. A structure-activity relationship study further expanded the inhibitor set to a third compound (dichlorophene) and each inhibitor was demonstrated to biochemically block autocatalytic crosslinking of FlgE from several pathogenic spirochetes with varied mechanisms and degrees of specificity. The most potent inhibitor, hexachlorophene, alters Lal crosslinking in cultured cells of Td and reduces bacterial motility in swimming plate assays. Overall, these results provide a proof-of-concept for the discovery and development of Lal-crosslink inhibitors to combat spirochete-derived illnesses.

biochemistry↗

bioRxiv2023

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

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 Treponema denticola (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 in vivo-derived samples from Treponema spp., Borreliella spp., Brachyspira spp., and Leptospira spp.. Like with Td, a mutant strain of the Lyme disease pathogen Borreliella burgdorferi unable to form the crosslink has impaired motility. FlgE from Leptospira spp. does not conserve the Lal-forming cysteine residue which is instead substituted by serine. Nevertheless, Leptospira interrogans 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 StatementThe 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 Treponema denticola 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. T. denticola and B. burgdorferi 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.

microbiology↗

bioRxiv2020

Molecular Mechanism for Rotational Switching of the Bacterial Flagellar Motor

The bacterial flagellar motor is a remarkable nanomachine that can rapidly rotate in both counter-clockwise (CCW) and clockwise (CW) senses. The transitions between CCW and CW rotation are critical for chemotaxis, and they are controlled by a signaling protein (CheY-P) that interacts with a switch complex at the cytoplasmic side of the flagellar motor. However, the exact molecular mechanism by which CheY-P controls the motor rotational switch remains enigmatic. Here, we use the Lyme disease spirochete, Borrelia burgdorferi, as the model system to dissect the mechanism underlying flagellar rotational switching. We first determined high resolution in situ motor structures in the cheX and cheY3 mutants in which motors are genetically locked in CCW or CW rotation. The structures showed that the CheY3 protein of B. burgdorferi interacts directly with the FliM protein of the switch complex in a phosphorylation-dependent manner. The binding of CheY3-P to FliM induces a major remodeling of the switch protein FliG2 that alters its interaction with the torque generator. Because the remodeling of FliG2 is directly correlated with the rotational direction, our data lead to a model for flagellar function in which the torque generator rotates in response to an inward flow of H+ driven by the proton motive force. Rapid conformational changes of FliG2 allow the switch complex to interact with opposite sides of the rotating torque generator, thereby facilitating rotational switching between CW and CCW.

microbiology↗