Elucidating the Bacterial and Host Mechanisms Governing B. burgdorferi-Related Type I Interferon Responses

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PROJECT SUMMARY B. burgdorferi (Bb), the vector-borne etiologic agent of Lyme disease, elicits robust inflammatory responses while evading and modulating host immunity to establish a multi-systemic infection. Bb isolates that differ in the presence or sequence of the 36kb linear plasmid (lp36) possess varied ability to disseminate and cause Lyme-related pathology, which has been linked to differential induction of host inflammatory and type I interferon (IFN-I) responses. Multiple innate immune receptors have been proposed to trigger IFN-I during infection; however, the exact host pathways governing Bb-dependent IFN-I production, the kinetics of IFN-I responses in infected tissues, and the Bb factors modulating IFN-I induction remain unknown. Ongoing studies have identified a novel role for the intracellular cGAS-STING DNA sensing pathway in controlling IFN-I induction during Bb infection. This proposal will therefore test the hypothesis that cGAS-STING and B. burgdorferi lp36 genes control the IFN-I response to shape infection kinetics and Lyme-related immunopathology. Aim 1 will employ a variety of human and mouse knockout and knockdown cells to characterize cGAS- and STING-dependent IFN-I production over the course of Bb infection. Next, high resolution microscopy and a cGAS immunoprecipitation-deep sequencing approach will be utilized to define the intracellular niches where cGAS senses Bb and to identify the bacterial and/or host DNA species that activate cGAS. To define roles in vivo, wildtype, cGAS-, and STING-deficient mice will be infected with bioluminescent Bb for spatiotemporal evaluation of borrelial load by an In Vivo Imaging System (IVIS). Finally, Aim 1 will employ a novel, IFN-I GFP reporter mouse strain to characterize the in vivo kinetics and cellular sources of interferon over a 35 day time course. Aim 2 will elucidate the role of Bb 36kb linear plasmid (lp36) and genes encoded in the bbk35-bbk50 region of lp36 in host IFN-I induction. Bb lacking lp36 entirely or the bbk35-bbk50 region, as well as Bb lacking subregions of bbk35-bbk50, will be assessed for their ability to adhere, be taken up, and/or trigger IFN-I by primary murine and human macrophages and fibroblasts. Furthermore, in vivo imaging by IVIS will be used to determine the dissemination phenotypes of all lp36 mutants and associated complement strains. The ability of mutant strains to induce Lyme-related immunopathology in mice will be examined over time. This work is innovative as it will be the first to define how Bb, a predominately extracellular pathogen, induces IFN-I through the intracellular cGAS-STING pathway. Moreover, it will characterize novel roles for candidate borrelial genes in triggering IFN-I during early and late phases of infection and will examine their contribution to Lyme disease pathogenesis. In the long term, this research has the potential to reveal new immunotherapeutic avenues that may be effective against active infection or persistent symptoms of B. burgdorferi, such as Lyme arthritis.