Functional and Mechanistic Studies of the Role of Lp17-encoded Factors in Host Adaptation by the Lyme Disease Spirochete - 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 pathogen in nature is completely dependent on its enzootic life cycle involving both a tick and reservoir host. The transition between these two very different host types requires the ability to rapidly adapt through changes in gene expression. Recent studies in our lab have provided evidence of the importance of lp17-resident genes for plasmid copy number control and resultant gene dosage effects that may have significant roles in host adaptation by B. burgdorferi. Despite this advance, there remains a fundamental gap in our understanding of the functional and mechanistic aspects of currently identified lp17-encoded factors. The overall objective of this application is to establish the functional outcome of lp17 copy number on gene dosage effects that correlate to the ability of the pathogen to adapt to the host environment. Additionally, we aim to determine that mechanism of action by the regulatory sRNA of lp17, SR0726, during host infection. Based on published and preliminary data, the central hypothesis is that bbd21-22 are important for proper copy number control of multiple B. burgdorferi plasmids that result in dosage effects of lp17-resident genetic factors important for host adaptation and tissue colonization. The rationale for the proposed research is that the expected outcomes will significantly advance our knowledge of the role that lp17 genes play in host adaptation by B. burgdorferi, and could ultimately lead to the identification of potential targets for the development of a vaccine and/or therapeutics against human infection. 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 our preliminary data, our hypothesis will be tested by pursuing two specific aims: 1) Establish the importance of bbd21 and bbd22 for copy number control of B. burgdorferi plasmids and gene dosage effects, and 2) Determine the regulatory mechanism of SR0726 during host infection. Under the first aim, the effects of bbd21 and/or bbd22 deletion on copy number control of plasmids other than lp17 will be examined, the ability of BBD21 and BBD22 to interact assessed, and the capacity of BBD21 and/or BBD22 to specifically bind DNA regions of multiple plasmids established. In the second aim, the approaches used will include RIL-seq analysis of SR0726 binding to target mRNA, mutational analysis of predicted complementarity binding regions of SR0726, and delineation of the upstream region necessary for regulated expression of SR0726. The proposed work is innovative because it involves the first mutational analysis of these lp17- resident genes in an infectious background strain to assess their mechanistic importance for both plasmid maintenance and gene regulation. When applied, the knowledge gained from the proposed studies have the potential to elucidate new drug targets to treat and prevent Lyme disease.
