Spirochetes are a medically significant but a remarkable poorly understood group of bacteria. These organisms cause a variety of important diseases including syphilis, Lyme disease, relapsing fever, and leptospirosis. They are also implicated in periodontal disease and human diarrheal disease. The present proposal aims to answer certain questions relating to the structure of spirochete periplasmic flagella (PFs) and motility. Spirochete PFs are the most complex of any bacterial flagellar filament. On the surface of the PFs is a protein sheath comprised of one to two FlaA protein species. The core is comprised of a polymer of three to four protein species referred to as FlaB proteins. The function of the individual PF proteins is not understood. To better understand the roles of these proteins in PF structure and motility, the applicant proposes to knock-out several genes involved in PF synthesis. The model system being used is Serpulina hyodysenteriae, formally known as Treponema hyodysenteriae. S. hyodysenteriae is the only spirochete species in which specific gene inactivation has been achieved. These spirochetes have PFs very similar to those found in Treponema pallidum, the causative agent for syphilis. T. pallidum still cannot be continuously cultured, let alone be manipulated for mutant analysis. Thus, because the PFs of both organisms are so similar, the results achieved with S. hyodysenteriae will be relevant to T. pallidum PF structure and motility. The applicant already has constructed three PF mutants, flaA1, flaB1, flaAlflaB1 by electroporation and allelic exchange. They hypothesize that these mutants have altered PF structure and that the altered PFs result in inefficient swimming. To test this, they will analyze PF structure and motility of the mutants, and make comparisons to the wild-type. To augment the above experiments, they will construct and analyze other motility mutants. These mutants include allelic exchange mutants in both the flaB2 gene, and the flgE gene. The latter gene encodes the hook gene, and mutations in this gene should result in cells completely deficient in PFs. The results obtained should lead to a clear understanding of the function of these proteins in PF structure and motility. The experiments proposed are the first to examine spirochete motility using a systematic molecular- genetic approach. They are especially relevant in that flagella and/or motility are implicated as a virulence factor for spirochetes and several other species of pathogenic bacteria.
