Mechanistic and Functional Analysis of a Putative Regulatory Factor in the Lyme Disease Spirochete

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Summary The ability of Borrelia burgdorferi to cause Lyme disease is highly dependent on its capacity to establish a successful infection upon entering the mammalian host. Moreover, survival of the Lyme disease pathogen in nature is completely dependent on its enzootic life cycle involving both a tick vector and reservoir host. The transition between these two very different environments requires the ability to rapidly adapt through changes in gene expression. Recent studies in our lab have provided evidence that the functional product of the bbd07 intergenic region of lp17 acts as a small non-coding RNA (sRNA) important for host adaptation. Despite this advance, there remains a fundamental gap in our understanding of the mechanistic aspects of bbd07-mediated gene regulation, and the genes subjected to its regulatory effects are unknown. Our long-term goals are to identify and characterize factors necessary for B. burgdorferi adaptation to the tick and mammalian host environments. The overall objective of this application is to establish the importance of bbd07 for regulation of genes important for host colonization by B. burgdorferi. Based on preliminary data, the central hypothesis of this proposal is that bbd07 acts as a sRNA essential for regulation of genes known to be important for host adaptation. The rationale for the proposed research is that bbd07 RNA and the gene products under its regulatory influence represent potential targets for the development of therapeutics against human infection by the pathogen, as well as disruption of its enzootic life cycle. 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 published and preliminary data, our hypothesis will be tested by pursuing the following two specific aims: 1) Determine the regulatory mechanism of bbd07 and identity of additional target genes, and 2) Establish a correlation between bbd07 expression and ospC repression during host infection. Under the first aim, the 5’ end of bbd07 RNA will be characterized by RACE, sRNA-mRNA target interactions will be mapped, and iTRAQ analysis will be used to determine the large-scale effects of bbd07 deletion on the B. burgdorferi proteome. Under the second aim, the relative levels of bbd07 and ospC expression in the murine and ticks hosts will be quantified, and OspC production visualized using an immunofluorescence assay. The proposed work is innovative because it utilizes a novel RNA-seq-based method to globally map sRNA-mRNA target interactions in B. burgdorferi, and is the first mutational analysis of bbd07 to assess its respective importance for regulation of genes involved in host adaptation. When applied, the knowledge gained from the proposed studies has the potential to elucidate new drug targets to treat and prevent Lyme disease.