Abstract Bacterial protein secretion is a fundamental physiological process that generates the cell envelope and maintains its integrity throughout the bacterial life cycle. In bacterial pathogens, a variety of protein secretion systems have been shown to deploy important virulence factors to the bacterial surface, into the milieu, or even directly into eukaryotic cells or other bacteria. Borrelia spirochetes, the causative agents of tick-borne Lyme disease and relapsing fever, have a unique double-membrane envelope with periplasmic flagella. The Borrelia surface lacks lipopolysaccharide and is instead covered by abundant, immunodominant and serotype-defining surface lipoproteins that serve as linchpins for transmission and pathogenesis. A recent study has shown that two thirds of the about 130 lipoproteins expressed by the Lyme disease bacterium Borrelia burgdorferi localize to the surface. Therefore, B. burgdorferi is a perfect model organism for investigations into the secretion of bacterial surface lipoproteins. Several seminal studies have demonstrated that (i) Borrelia surface lipoprotein secretion determinants commonly localize to N-terminal disordered tether regions of the mature lipoproteins, (ii) translocation through the outer membrane can initiate at a lipoprotein’s C terminus and requires an at least partially unfolded conformation, and (iii) Borrelia surface lipoproteins are ultimately anchored in the surface leaflet of the outer membrane bilayer. These data support the hypothesis that the Borrelia surface lipoprotein secretion pathway includes a periplasmic mechanism that prevents premature folding of surface lipoprotein and an outer membrane translocon complex that allows for the flipping of lipoproteins from the periplasm to the surface. This proposal will test the above hypothesis by identifying and mechanistically defining the components of the B. burgdorferi surface lipoprotein secretion pathway. Aim 1 will build on recent CRISPRi gene silencing data and employ site-directed mutagenesis and quantitative proteomics to begin determining the structure- function relationships of a B. burgdorferi outer membrane protein shown to facilitate translocation of lipoproteins from the periplasm to the surface. Fluorescent tags will be used to further define the sequestration of the B. burgdorferi lipoproteome in space and time. Aim 2 will use the same approaches as well as X-ray crystallography to characterize periplasmic proteins that are hypothesized to release surface lipoproteins from the inner membrane and deliver them to the outer membrane. Together, these experiments will use novel approaches to further elucidate how emerging pathogens of global importance generate and maintain their interface with the host. This will ultimately yield better tools for diagnostics and improved strategies for prevention and treatment.