ELUCIDATING MECHANISMS FOR PERSISTENT INFECTION BY THE RELAPSING FEVER SPIROCHETE BORRELIA HERMSII
James, Allison Eavey
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Relapsing fever is a tick-borne bacterial disease caused by a number of species in the genus Borrelia. Persistence is a key component of the lifecycle of relapsing fever Borrelia as it improves the chance for tick acquisition and subsequent transmission. During infection of the mammalian host, persistence is achieved through antigenic variation, where the pathogen sequentially changes its immunodominant outer surface lipoprotein to evade the host’s immune response. Correspondingly, this antigenic variation system is directly responsible for the episodic febrile events characteristic of relapsing fever. Besides the antigenic variation system, no other factors required for B. hermsii persistence have been identified. In this dissertation, two distinct, putative determinants for B. hermsii persistence were investigated: a DNA adenine-specific methyltransferase gene (dam), and two cis-acting components of the within-host antigenic variation system. Because Dams in other pathogens have a demonstrated role in pathogenesis and persistence, a putative dam of B. hermsii was investigated for its requirement in infectivity and persistence. The results of this study are reported in chapter two. First, the putative B. hermsii dam locus was verified as a Dam. The locus in B. hermsii was then disrupted through allelic exchange, generating a dam- B. hermsii strain. After verifying the absence of Dam function, it was determined that the mutant was infectious and persistent in immunocompetent mice. Overall, findings from this study verified that dam is not a determinant for B. hermsii persistence. In chapter three, two cis-acting DNA elements, the upstream homology sequence (UHS) and downstream homology sequence (DHS), were investigated for their role in the gene conversion event responsible for antigenic variation. Employing a targeted deletion-in cis complementation technique that has not previously been applied to any relapsing fever Borrelia spp., the UHS and DHS elements were genetically mutated in B. hermsii. Murine infection with the mutants revealed that that the UHS and a portion of the DHS are required for antigenic variation due to the observed lack of persistence in immunocompetent mice. These are the first studies to establish the importance of the UHS and DHS using a direct, mutational approach.