Mechanistic Studies of the Role of Genetic Factors in VlsE Antigenic Variation by the Lyme Disease Spirochete - Project Summary A key mechanism for immune evasion and persistent infection by the Lyme disease spirochete, Borrelia burgdorferi, is antigenic variation of the VlsE surface protein. The native vlsE gene has not been receptive to mutation by any available methods over the years, which has greatly impeded progress in the study of this system. Using an ectopically expressed copy of vlsE, we have strong evidence that vlsE recombination can occur in trans, and that mutation of individual genetic elements within the vlsE gene copy are critical for expression and gene conversion. Most importantly, this work led to the discovery of a novel method for introducing mutations into the native vlsE gene via a mutated trans copy harbored on a linear shuttle vector. Despite this important evidence, certain mechanistic aspects involved in vlsE expression and recombination remain unknown. Our long-term goals are to determine the mechanism of VlsE antigenic variation and overall implications of surface antigen protection promoted by the VlsE lipoprotein. The objective of this application is to decipher the mechanistic details of cis-acting factors involved in vlsE expression and gene conversion. Based on published studies and preliminary findings, our central hypothesis is that specific guanine-rich DNA regions represent cis-acting elements important for the gene conversion mechanism, and that the putative SpoVG binding site is necessary for vlsE expression and recombination. The rationale for the proposed research is that identifying the mechanistic details of this system will provide the knowledge required to design future long-term studies targeted at dissecting the overall mechanism. Together, the proposed research is relevant to NIH’s mission that pertains to developing fundamental knowledge that will potentially help to reduce the burdens of human illness and disability. Guided by preliminary findings, our hypothesis will be tested by pursuing two specific aims: 1) Establish the importance of guanine-rich DNA regions for vlsE recombination; and 2) Demonstrate a role for the putative SpoVG binding site in vlsE expression and gene conversion. Under the first aim, mutations of guanine-rich DNA sequences within vlsE will be generated in B. burgdorferi and used to infect both immunologically competent and deficient mice to look for loss of bacterial persistence due to a lack of VlsE antigenic variation compared to the wild type control. Under the second aim, qRT-PCR will be utilized to quantitate the relative vlsE expression levels during host infection by a SpoVG binding site mutant clone compared to a wild type control, and any effects on vlsE gene conversion assessed. The proposed work is innovative because it makes use of a newly discovered method to introduce genetic mutations into the native vlsE gene on the lp28-1 plasmid to address the novel concept that vlsE expression is required for the antigenic variation process. Overall, these studies will significantly advance our knowledge of immune evasion by B. burgdorferi, and provide more useful strategies to prevent and treat Lyme disease in humans.
