Mutational analysis of the vlp/vsp antigenic variation system of the relapsing fe

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DESCRIPTION (provided by applicant): A key mechanism for immune evasion and recurrent bacteremia by the East African relapsing fever spirochete, Borrelia duttonii, is antigenic variatio of the Vlp and Vsp surface proteins. Previous studies involving DNA sequence analysis of Borrelia hermsii serotypes, the species endemic to the western United States, have implicated the importance of an upstream homology sequence (UHS) and downstream homology sequence (DHS) for antigenic switching. Although DNA sequence and statistical analysis has implicated the importance of these DNA elements for vlp/vsp antigenic switching, direct mutational studies providing a mechanistic role for these elements in antigenic variation by any relapsing fever Borrelia species is still lacking. Our long-term goals are to decipher the mechanistic details of vlp/vsp antigenic variation in B. duttonii, and to expand these findings to the louse-borne variant, B. recurrentis. The objective of this application is to verify putative DN elements of B. duttonii that are required for antigenic variation. Our central hypothesis is that UHS and DHS sites function as cis-acting DNA elements that are necessary for efficient gene conversion at the vlp/vsp expression site. The rationale for the proposed research is that successful completion will demonstrate the practicality of our experimental approach, which is necessary in order to obtain long-term funding for further research on this important immune evasion mechanism. 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. Guided by DNA sequence analysis and previously published work, our hypothesis will be tested by pursuing two specific aims: 1) Establish the importance of the UHS and DHS for efficient vlp/vsp recombination; and 2) Establish the requirement of a DHS-resident inverted repeat for vlp/vsp recombination. Under the first aim, mutations and deletions within the UHS and DHS elements will be generated. These mutants will then be used to infect immunocompetent mice to look for a loss of antigenic switching compared to wild-type controls. Under the second aim, the inverted repeat within the DHS will be interrupted while keeping the overall sequence length the same. Antigenic variation compared to wild-type controls will be monitored after infecting immunocompetent mice. The proposed work is innovative, because it represents the first time that an antigenic variation system of any relapsing fever Borrelia species has been targeted for mutation. When applied, these results are expected to allow the targeting of this system in order to significantly reduce the ability of this pathogen to persist ad cause disease in the mammalian host.