Academic, Borrelia burgdorferi, purine transport, ixodes scapularis, tick, lyme disease, mouse infection, competation assay, hypoxanthine, adenine, guanine
Lyme disease, the most common tick borne disease in United States, is caused by the bacterial pathogen Borrelia burgdorferi. In nature, B. burgdorferi exists in an enzootic infectious cycle between an arthropod vector and mammalian hosts. Identification and characterization of the genes essential for B. burgdorferi survival throughout its infectious cycle is an important step toward understanding the molecular mechanisms involved in B. burgdorferi pathogenesis. B. burgdorferi contains a small genome, which lacks the genes encoding for the enzymes required for de novo synthesis of amino acids, fatty acids and nucleic acid precursors. Therefore, the spirochete is dependent upon the host environment for the uptake of these essential nutrients. Purines are required for the synthesis of nucleotides for the biosynthesis of DNA and RNA. Due to the lack of de novo purine synthesis, the ability of B. burgdorferi to salvage purines from its host environments is essential to its survival. While the enzymes critical for the B. burgdorferi purine salvage pathway are known, the transporters involved in the uptake of purines from the host environments are not. The work in this thesis is focused on identification of the genes encoding purine permeases in B. burgdorferi and genetic and biochemical characterization of their functions in the infectious cycle of B. burgdorferi. Here, we demonstrate that homologous genes bbb22 and bbb23 present on circular plasmid 26 encode for purine permeases, which are important for transport of hypoxanthine, adenine and guanine. Furthermore, genes bbb22-23 together were essential for B. burgdorferi infection in mice. BBB22 and BBB23 share 78% amino acid identify. And although, individually both BBB22 and BBB23 were found to be capable of purine transport, BBB22 has higher affinity for hypoxanthine and adenine compared to BBB23. Moreover, the bbb22 gene alone was sufficient to restore mouse infectivity to spirochetes lacking both bbb22 and bbb23, whereas, bbb23 was not. Nonetheless, the spirochete loads in the tissues of mice infected with B. burgdorferi carrying bbb22 alone were significantly reduced compared to B. burgdorferi carrying both bbb22 and bbb23, demonstrating the importance of the two genes together for the spirochetes to achieve wild type levels of infection. In ticks, genes bbb22 and bbb23 were dispensable for spirochete survival but contributed to spirochete replication in fed larvae. The replication of spirochetes lacking bbb22-23 in larval ticks was restored to wild type levels by the reintroduction of the low affinity purine transporter encoded by bbb23 alone. Overall, we have identified a purine transport system in B. burgdorferi, which is essential for spirochete survival in the mammalian host and contributes to spirochete replication in the tick vector. As B. burgdorferi lacks typical virulence factors and toxins, these studies highlight the critical role of physiological functions in the virulence of this pathogen. Moreover, the BBB22-23 in vivo essential transport system may represent a novel therapeutic target to deliver antimicrobial drugs to treat Lyme disease.
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Doctor of Philosophy (Ph.D.)
College of Medicine
Molecular Biology and Microbiology
Length of Campus-only Access
Doctoral Dissertation (Open Access)
Dissertations, Academic -- Medicine; Medicine -- Dissertations, Academic
Jain, Sunny, "Genetic and biochemical characterization of the roles of two putative purine transporters in the infectious cycle of Borrelia burgdorferi" (2014). Electronic Theses and Dissertations, 2004-2019. 4671.