Biology subjects

Lynch, M. J.

Publications and source records attributed to Lynch, M. J..

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↗

bioRxiv2024

MCP5, a methyl-accepting chemotaxis protein regulated by both the Hk1-Rrp1 and Rrp2-RpoN-RpoS pathways, is required for the immune evasion of Borrelia burgdorferi

Borrelia (or Borreliella) burgdorferi, the causative agent of Lyme disease, is a motile and invasive zoonotic pathogen, adept at navigating between its arthropod vector and mammalian host. While motility and chemotaxis are well established as essential for its enzootic cycle, the function of methyl-accepting chemotaxis proteins (MCPs) in the infectious cycle of B. burgdorferi remains unclear. In this study, we demonstrate that MCP5, one of the most abundant MCPs in B. burgdorferi, is differentially expressed in response to environmental signals as well as at different stages of the pathogens enzootic cycle. Specifically, the expression of mcp5 is regulated by the Hk1-Rrp1 and Rrp2-RpoN-RpoS pathways, which are critical for the spirochetes colonization of the tick vector and mammalian host, respectively. Infection experiments with an mcp5 mutant revealed that spirochetes lacking MCP5 could not establish infections in either C3H/HeN mice or Severe Combined Immunodeficiency (SCID) mice, which are defective in adaptive immunity, indicating the essential role of MCP5 in mammalian infection. However, the mcp5 mutant could establish infection and disseminate in NOD SCID Gamma (NSG) mice, which are deficient in both adaptive and most innate immune responses, suggesting a crucial role of MCP5 in evading host innate immunity. In the tick vector, the mcp5 mutants survived feeding but failed to transmit to mice, highlighting the importance of MCP5 in transmission. Our findings reveal that MCP5, regulated by the Rrp1 and Rrp2 pathways, is critical for the establishment of infection in mammalian hosts by evading host innate immunity and is important for the transmission of spirochetes from ticks to mammalian hosts, underscoring its potential as a target for intervention strategies. SUMMARYLyme disease is the most commonly reported arthropod-borne illness in the US, Europe, and Asia. The causative agent of Lyme disease, Borrelia burgdorferi, is maintained in an enzootic cycle involving arthropod vectors (Ixodes ticks) and rodent mammalian hosts. Understanding how B. burgdorferi moves within this natural cycle is crucial for developing new strategies to combat Lyme disease. The complex nature of the enzootic cycle necessitates sensory-guided movement in response to environmental stimuli. B. burgdorferi possesses a unique and intricate chemotaxis signaling system, with methyl-accepting chemotaxis proteins (MCPs) at its core. These proteins are responsible for sensing environmental signals and guiding bacterial movement toward or away from stimuli. This study found that one of the MCPs, MCP5, is highly expressed and differentially regulated during the enzootic cycle by the Hk1-Rrp1 and Rrp2-RpoN-RpoS pathways. MCP5 is crucial for mammalian infection, aiding in immune evasion and transmission from ticks to mammals, providing a foundation for further research into B. burgdorferis navigation within its hosts.

microbiology↗

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↗