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Characterization of the Unique Flagellar Hook Structure of the Spirochetes Borrelia burgdorferi and Treponema denticola Kelly Ann Heintzelman Miller The long-term goal of this project is to better understand Borrelia burgdorferi and Treponema denticola motility as it relates to pathogenesis. The structure of the spirochete periplasmic flagella is unique when compared to externally located flagella. One unusual characteristic is the flagellar hook, which serves as a universal joint coupling rotation of the membrane bound motor complex to the flagellar filament. The hook is composed of approximately 120 FlgE monomers, and in bacteria with externally located flagella, these structures readily disassociate into monomers (~50 kDa) when treated with heat and detergent. However, in spirochetes the FlgE monomers form a large mass of over 250 kDa. We hypothesize that this high molecular weight complex (HMWC) is the result of covalent cross-linking of FlgE proteins. Cross-linking of bacterial flagella proteins has not been examined previously. Our goals are to thoroughly characterize the cross-linking in the B.burgdorferi and T. denticola hook proteins. We anticipate that once we have this information, new drugs could be developed to inhibit this cross-linking, which we anticipate will severely affect the spirochetes' motility and virulence capability. Thus, the proposed project’s long-term goal is centered on developing new drug treatments for Lyme disease as well as other spirochetal diseases, including syphilis. We found that the HMWC of both B. burgdorferi and T. denticola are stable to a variety of denaturants and is not an artifact of boiling. Previously, western blot analysis was the sole indication that spirochetes crosslink their flagellar hook proteins. By making several modifications to the published procedure for isolating periplasmic flagella, I was able to obtain enough hook protein of both B. burgdorferi and T. denticola for mass spectrometry analysis. The mass spectrometry analysis confirmed that the spirochete HMWC is composed of FlgE, identified FlgE as the sole protein in these complexes, and that in specific regions the mass spectrometry patterns are different for the monomer and HMWC. We observed that T. denticola FlgE over-expressed in Escherichia coli forms a stable HMWC in vitro, and used the in vitro HMWC to study the effects of both chemical treatments and point mutations on HMWC formation. Finally, I propose a mechanism of cross-linking that is consistent with the mass spectrometry, biochemical, and mutational analyses. We hypothesize that FlgE cross-linking strengthens the hook structure for optimal spirochete motility.