PROBING THE LIPOPROTEIN SECRETION PATHWAY IN BORRELIA BURGDORFERI

Abstract

The bacterial agent Borrelia burgdorferi causes Lyme disease, a debilitating inflammatory disease and a major public health issue in the United States and around the world. Early studies of B. burgdorferi pathogenesis determined the major immunogenic factors to be lipid-modified proteins (lipoproteins). The B. burgdorferi genome encodes more lipoproteins than any other organism studied to date. The majority of these lipoproteins are tethered to the B. burgdorferi cell surface, creating the medically important host-pathogen interface. In addition, many studies have revealed the significance of surface lipoproteins to the B. burgdorferi life cycle in both the tick vector and mammalian host. Despite the importance of lipoproteins, little is known about the protein machinery and molecular events responsible for lipoprotein transport to the borrelial outer surface. The goal of this dissertation is to shed light on the mechanism of lipoprotein trafficking in B. burgdorferi by (i) investigating borrelial homologs of known lipoprotein transport machinery (the Lol system), (ii) localizing a known lipoprotein, and (iii) investigating the Borrelia surface proteome to identify substrates and factors involved in lipoprotein transport. First, we used genetic manipulation and biochemical analyses to understand the role of Lol pathway homologs in surface and sub-surface lipoprotein transport. Our experiments indicate that the Lol pathway does not play a primary role in transport of surface lipoproteins. Nevertheless, a block in the pathway does impact localization of subsurface lipoproteins as well as OM porins. Second, we demonstrated inner membrane localization of IpLA7, a major immunogenic protein in Lyme disease. Of the many predicted lipoproteins in the borrelial genome, few have been localized to the periplasm. Third, we constructed reporter surface lipoproteins to identify interacting proteins along the transport pathway and tested several methods to characterize the outer membrane protein composition. Finally, we used in silico prediction algorithms to identify novel candidate integral outer membrane proteins, which may be involved in lipoprotein transport. Together, these data contribute to characterizing the molecular events responsible for subsurface and surface outer membrane lipoprotein secretion in B. burgdorferi, an endeavor vital to understanding the regulation and processing of these key virulence factors and thus the pathogenesis of Lyme disease.