DELINEATION OF UNIQUE FLAGELLAR PROTEINS IN SPIROCHETES

Spirochetes are a group of poorly studied medically significant bacteria. The motility of spirochetes is driven byperiplasmic flagella, which reside and rotate within the periplasmic space. These bacterial motility is crucial forvirulence by all pathogenic spirochetes studied to-date including Borrelia burgdorferi. While motility plays sucha vital role, we know very little about the periplasmic flagellar assembly, operation or organization in anyspirochete. Although many components of the periplasmic flagellum have highly conserved counterparts in theexternal flagella from the model organisms Salmonella enterica and Escherichia coli, some unique componentsof the periplasmic flagella clearly distinguish them from external flagella. Most importantly, our recent studieshave provided the first evidence that the novel spirochete-specific component known as the periplasmic collaris essential for flagellar assembly and orientation, the distinctive morphology and motility of these bacteria.However, very little is known about the genes encoding the periplasmic collar or their function in anyspirochete. Based on our preliminary data, we hypothesize that the periplasmic collar is comprised of multiplenovel spirochete-specific proteins, and that each of these proteins plays a distinct role in flagellar assembly,spirochetes distinctive morphology and motility. To address this hypothesis, we propose two specific aims.Aim 1 is to identify the proteins that make-up the large collar complex, determine their native cellular structureand function in B. burgdorferi. Moreover, to extend the relevance of B. burgdorferi periplasmic collar studies,we propose to determine if these novel flagellar proteins or their function is conserved in other spirochetes.Aim 1 is expected to be accomplished by using bioinformatics, genetics, various biochemical assays and cryo-electron tomography. We expect to identify the proteins encoding the collar complex structure, their function,location, structure, and assembly in the spirochetes. Aim 2 is proposed to determine the sequential assemblyof the periplasmic collar proteins in the cell envelope and to understand the interactions of novel collar proteinswith other prominent flagellar proteins and their impacts on increased torque required for the periplasmicflagella to rotate in viscous or complex medium such as the mammalian tissues. We plan to accomplish thisaim using various mutational, biochemical, and cryo-electron tomography. We expect to understand how theperiplasmic flagella are assembled in the spirochete and their overall impacts in B. burgdorferi. The knowledgegained from this project is fundamental to understand the biosynthesis, assembly, and function of the novelflagellar proteins not only in B. burgdorferi and Leptospira but also in other medically significant yetuncultivable spirochetes such as Treponema pallidum. These studies can also lead to applications in structure-based drug design to disrupt motor assembly, therefore blocking the spread of Lyme as well as otherspirochete-borne diseases.