Transendothelial migration of the Lyme disease spirochete involves spirochete internalization as an intermediate step through a transcellular pathway that involves Cdc42 and Rac1

Despite its importance in pathogenesis, the hematogenous dissemination pathway of <i>Borrelia burgdorferi</i> is still largely uncharacterized. To probe the molecular details of transendothelial migration more easily, we studied this process using cultured primary or telomerase-immortalized human microvascular endothelial cells in a medium that maintains both the human cells and the spirochetes. In <i>B. burgdorferi</i>-infected monolayers, we observed ~55% of wild-type spirochetes crossing the monolayer. Microscopic characterization revealed entrance points across the cellular surface rather than at cellular junctions, supporting a transcellular route. In support of this pathway, locking the endothelial junctions using a vascular endothelial protein tyrosine phosphatase (VE-PTP) inhibitor did not reduce transendothelial migration. We also used inhibitors to block the most common endocytic pathways to elucidate effectors that might be involved in <i>B. burgdorferi</i> uptake and/or transmigration. Directly inhibiting Cdc42 reduced spirochete transmigration by impeding internalization. However, blocking Rac1 alone dramatically reduced transmigration by ~84% and resulted in a concomitant doubling in spirochete accumulation in the cell. Our combined results support that <i>B. burgdorferi</i> internalization is an intermediate step in the transendothelial migration process, which requires both Cdc42 and Rac1; Cdc42 is needed for spirochete internalization, while Rac1 is required for cellular egress. These are the first two host proteins implicated in <i>B. burgdorferi</i> transmigration across endothelial cells.IMPORTANCELyme borreliosis is caused by <i>Borrelia burgdorferi</i> and related bacteria. It is the most common tick-transmitted illness in the Northern Hemisphere. The ability of this pathogen to spread to a wide variety of locations results in a diverse set of clinical manifestations, yet little is known regarding vascular escape of the spirochete, an important pathway for dissemination. Our current work has studied the traversal of <i>B. burgdorferi</i> across a monolayer of microvascular endothelial cells grown using a new culture system. We show that this occurs by passage of the spirochetes directly through cells rather than at cellular junctions and that internalization of <i>B. burgdorferi</i> is an intermediate step in transmigration. We also identify the first two host proteins, Cdc42 and Rac1, that are used by the spirochetes to promote traversal of the cellular monolayer. Our new experimental system also provides a new avenue for further studies of this important process.