Foor, Shelby DimityLyme Disease (LD) is a tick-borne disease caused by a group of gram-negative-like spirochetal pathogens called Borrelia burgdorferi sensu lato. The number of cases reported in the United States have dramatically increased with CDC estimating 476,000 cases annually. This multifaceted infection can spread throughout the entire body, causing clinical complications of the central nervous system, joint and heart. Early antibiotic treatment is available and effective; however, untreated patients can develop chronic symptoms, and even after antibiotics, symptoms of unknown etiology and pathogenesis can develop into post-treatment Lyme disease syndrome (PTLDS). The enzootic life cycle of B. burgdorferi is maintained typically between a small rodent and the Ixodes tick vector, where transmission occurs during tick feeding on a host. Infection establishes after B. burgdorferi is deposited in the dermis and undergoes the required shift in its protein expression profile necessary to support spirochete persistence and pathology, often highlighting protein targets for development of diagnostic, therapeutic, and preventative measures. Two such proteins identified, BB0238 and BB0323, serve as novel virulence determinants and are essential for mammalian infection. These two proteins directly interact, mutually stabilize each other post-translationally, and form an essential complex required for infection; however, their precise functions remain undetermined. In collaborative efforts, we predicted a two-domain structure of BB0238. The N-terminal domain was predicted by AI methods to harbor an antiparallel helix-turn-helix motif (HTH) followed by a third helix and a low-confidence predicted meandering segment. The C-terminal domain structure was determined by X-ray crystallography as well as predicted with high confidence to adopt an α+β fold that resembles closely that of the nuclear transport factor 2 (NTF2) superfamily. While full-length BB0238 lacks homology to singular proteins of known functions, the individual N- and C-terminal regions display structural homology to non-bacterial proteins, particularly to eukaryotic sorting, or transport proteins, suggesting that BB0238 supports an unconventional function in spirochetes. We discovered that BB0238 binds another borrelial protein annotated as BB0108, orthologs of two bacterial chaperones and foldases, the extracellular membrane anchored PrsA, and the periplasmic SurA. This identified interaction requires further investigation, however, may be important for BB0238 protein stability or assist with the novel BB0238 function discovered herein, which regulates proteolytic processing of BB0323. Furthermore, We show that key amino acid residues within the HTH stabilize BB0238 in an environment-specific manner, influence its oligomerization properties, and facilitate tick-to-mouse transmission by aiding spirochete evasion of host cellular immunity, underscoring BB0238’s ability to support microbial establishment during early mammalian infection. Together, these studies highlight the divergent evolution of multidomain spirochete proteins involved in multiplex protein-protein interactions, possibly facilitating multiple functions, which support pathogen survival and thus, represent novel targets for vaccine and therapeutic development against Lyme disease.enDissertationMolecular biologyPathologyImmunologyBorrelia burgdorferiLyme DiseaseProtein-Protein InteractionVirulence Factor