Lyme disease is an infection caused by a spirochete, Borrelia burgdorferi, that is inoculated into the skin by a tick vector in the Ixodes ricinis/Ixodes persulcatus species complex. After inoculation in the skin spirochetes spread centripetally resulting in a characteristic erythematous lesion called erythema migrans. Subsequently, the organisms disseminate widely resulting in a clinical syndrome which principally involves the central nervous system, heart, diarthrodial joints and the skin. For virtually all bacteria which disseminate from a skin or soft tissue inoculation site bacterial proteases, which digest extracellular matrix proteins, facilitate spreading in the skin and subsequent invasion into the lymphatic or vascular circulations. We have found that B. burgdorferi lacks these proteases but is able to spread from its inoculation site in the skin. Instead, B. burgdorferi has evolved a newly identified mechanism for accomplishing this step in pathogenesis by utilizing human proteases which are generated at the inoculation site and become bound to the bacterial surface. More specifically, at the vascular injury site created by the tick vector, B. burgdorferi subverts the host's fibrinolytic system by binding human urokinase type plasminogen activator (uPA) and human plasminogen (Pgn) which generates bioactive human plasmin on the surface of the spirochete. Human plasmin bound to B. burgdorferi is a highly stable, non-immunogenic, potent serine protease with broad substrate specificity including extracellular matrix and basement membrane components. When human uPA is bound to the surface of B. burgdorferi, the number of spirochetes required to establish an infection in mice inoculated via the intraperitoneal route is reduced by a least 1,000 fold. In this proposal we will investigate aspects of the biochemical, morphologic, biologic and immunologic consequences of host fibrinolytic protein binding to B. burgdorferi. First, we will characterize and define the membrane binding sites for human uPA and plasminogen/plasmin on the spirochete surface. In conjunction with these biochemical studies we will use confocal, scanning and transmission electron microscopy to investigate the microanatomy of uPA and Plg binding to B. burgdorferi and other borrelia species. We will also investigate the hypothesis that during tick repletion with host blood, fibrinolytic proteins are generated at the vascular injury site which are imbibed and then become bound to spirochetes in the midgut of the vector thereby generating surface bound plasmin and facilitating invasion and dissemination of B. burgdorferi in the tick. Finally we will study the humoral immune response of patients with Lyme disease to the spirochete binding sites for human fibrinolytic proteins, determine whether spirochetes which have uPA, plasminogen, or plasmin bound to their surface preferentially localize to sites of vascular injury and whether uPA and Plg binding to B. burgdorferi enhances the invasiveness of spirochetes in vitro. These studies form the basis for a better understanding of the host-vector-pathogen interactions of Lyme disease and may identify new vaccine candidates.
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