Toward novel therapies against Lyme disease through the inhibition of lysinoalaine cross-linking in the bacterial flagella.

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Treponema pallidum (Tp), Borrelia burgdorferi (Bb), Leptospira interrogans (Li) and Treponema denticola (Td) are spirochete bacteria that cause syphilis, Lyme disease, leptospirosis, and are associated with periodontal diseases in humans, respectively. These organisms cause substantial morbidity and mortality in the United States and throughout the world. Owing to the prevalence of Lyme disease and emergence of antibiotic resistance in Tp and Td, our long-term goal is to develop novel drugs that specifically treat diseases caused by spirochetes. Spirochetes are highly invasive bacteria, and their unique mode of motility plays an essential role in their ability to penetrate and invade host tissues and organs. The flagella of spirochetes reside within the periplasm and are thereby shielded from the immune system. A key component of bacterial flagella termed the hook joins the flagella filament to the membrane-imbedded rotary motor. The hook consists of multiple FlgE proteins, and in contrast to other bacterial flagella, spirochete FlgE proteins are covalently cross-linked to one another. This cross-link involves formation of a novel lysinoalanine (Lal) amino acid. The central hypothesis is that the FlgE proteins are covalently cross-linked to strengthen the hook for optimal motility and virulence. It is proposed that understanding the structure of the cross-link, its chemical synthesis and its role in virulence will lead to the development of drugs that inhibit cross-linking for treating spirochetal diseases. Specific Aim 1. Investigate the effect of FlgE cross-linking on the infectivity of Bb. Mutants of Td and Bb that are unable to cross-link their hook proteins are also altered in shape and deficient in translational motility. To determine the importance of cross-linking for Bb virulence, we will produce a virulent strain impaired in FlgE cross-linking and evaluate its ability to swim and sustain infections in both mice and ticks. Specific Aim 2. Develop small molecule inhibitors of FlgE cross-linking. The chemistry of LA formation is biologically unprecedented. Based on mechanistic and structural studies we have established cross-linking assays with recombinant FlgE proteins from Td and Bb for large-scale inhibitor screens. With these assays we have discovered an inhibitor of FlgE cross-linking and Bb motility. We will further characterize the action of this compound and continue to identify and characterize additional classes of inhibitors to be used for studying pathogenesis in hosts and eventually as lead compounds for therapeutics. Specific Aim 3. Determine the effects of FlgE cross-linking on the structure and stability of the flagella hook. To test whether the FlgE cross-links stabilize the hook to resist the high mechanical stress it likely experiences in the periplasmic space, we will analyze the physical properties of cross-linked and non-cross- linked hooks. In addition, the requirement of cross-linking will be tested by chemically restoring function in absence of Lal.