Summary Borrelia hermsii is a causative agent of tick-borne relapsing fever, which is a bacterial illness characterized by severe recurrent febrile episodes. The undulating fever that is characteristic of the disease is a direct result of Vmp antigenic variation and sequential immune evasion by the pathogen. Recent work in our lab has provided evidence that a region upstream from the vmp locus may be involved in the regulation of Vmp expression, and that a mutant clone deficient in vmp expression was unable to undergo vmp recombination. Additionally, a mutant clone lacking an inverted DNA repeat residing in a downstream homology region did not exhibit Vmp antigenic variation. Despite these advancements, the exact identity and role of cis-acting genetic elements in vmp regulated expression and recombination remain unknown. The objective of our application will be to identify cis-acting DNA regions of the vmp locus that are important for vmp regulated expression and recombination. Based on published and preliminary findings, our central hypothesis is that the tandem inverted repeat sequences are involved in the regulated expression of vmp, and that vmp transcription is required for the gene conversion process. Additionally, we hypothesize that it is the secondary hairpin structure of the DHS- resident inverted repeat sequence that is essential for vmp recombination. The rationale for addressing these knowledge gaps is that the resulting outcomes will lay the groundwork for additional investigations aimed at disrupting the antigenic variation system that underpins the pathogenesis of disease. Thus, the proposed research is relevant to that part of NIH’s mission that pertains to developing fundamental knowledge that will potentially help to reduce the burdens of human illness and disability. Our central hypothesis will be tested by pursuing three specific aims: 1) Determine the role of the tandem inverted repeat sequences for regulated expression of vmp, 2) Determine the importance of vmp expression for gene conversion, and 3) Establish the requirement of the DHS-resident inverted repeat structure for vmp recombination. Under the first aim, individual inverted DNA repeats will be deleted using our telomere-mediated deletion/complementation technique. Transcription and translation of vmp from mutant clones or an isogenic wild type control will be determined via qRT-PCR and Western blot analysis, respectively. Additionally, mRNA transcript levels will be quantified from blood samples obtained from infected mice at varying time intervals and compared to those from the wild type control. Under the second aim, mutations will be generated within the vmp promoter region and used to infect mice to look for a loss of antigenic switching compared to a wild type control. In the third aim, the inverted repeat within the DHS will be interrupted or DNA sequence altered while retaining the overall sequence length. Antigenic variation compared to the wild type con...