Inhibitors of Lysinoalanine Cross-Linking in the Flagella Hook as Antimicrobials against Spirochetes

Area of Science:

• Microbiology
• Bacteriology
• Drug Discovery

Background:

• Spirochetes are invasive bacteria causing diseases like Lyme disease, syphilis, and leptospirosis.
• Their motility, essential for infection and immune evasion, relies on periplasmic flagella (PFs) containing lysinoalanine (Lal) cross-links.
• Lal cross-links are conserved in pathogenic spirochetes and are vital for motility in species like *Treponema denticola* and *Borreliella burgdorferi*.

Purpose of the Study:

• To identify inhibitors of lysinoalanine (Lal) cross-linking in spirochetes.
• To explore Lal cross-links as a potential therapeutic target for novel antimicrobials.
• To validate a high-throughput screening method for discovering such inhibitors.

Main Methods:

• Development and application of a NanoLuc-based high-throughput screen.
• Screening a library of clinically approved small molecules.
• Structure-activity relationship studies to expand the inhibitor set.
• Biochemical assays to assess FlgE cross-linking inhibition.
• Cell-based assays and motility assays in pathogenic spirochetes.

Main Results:

• Identified two structurally related Lal cross-link inhibitors, hexachlorophene and triclosan, from approved small molecules.
• A third inhibitor, dichlorophene, was identified through structure-activity relationship studies.
• Inhibitors demonstrated biochemical blocking of FlgE autocatalytic cross-linking in various spirochetes.
• Hexachlorophene, the most potent inhibitor, disrupted Lal cross-linking in *Treponema denticola* cells and reduced bacterial motility.

Conclusions:

• Lysinoalanine (Lal) cross-link inhibitors represent a promising new class of antimicrobials against spirochete-borne diseases.
• The developed NanoLuc-based screen is effective for discovering inhibitors of bacterial motility mechanisms.
• Targeting Lal cross-linking provides a viable strategy for combating pathogenic spirochetes.