Cryptococcal meningitis kills approximately 625,000 immunocompromised individuals each year, mainly in developing areas of the world. The pathogenic fungus responsible for this disease, Cryptococcus neoformans, enters the host by inhalation; how it specifically targets and invades the brain to cause fatal infection is a central question in the field. Several distinct routes of entry to the central nervous system (CNS) have been proposed and supported by experimental evidence, but gaps remain in mechanistic understanding of these routes. Our goal in this R21 application is to identify host endothelial factors that participate in cryptococcal traversal of the blood-brain barrier (BBB), as a step toward our long-term objective of defining the molecular components and specific roles of the pathways used by C. neoformans to enter the CNS. We have developed a high-throughput screening assay to measure the transit of free C. neoformans across model BBB that have been treated with siRNA. In Aim 1 we will use it to screen an siRNA library targeting endothelial surface proteins, and will validate hits from the screen for further study. In Aim 2 we will assess the specificity and roles in BBB transit of selected hits from Aim 1, using a combination of imaging, in vitro assays, and in vivo studies. While these aims are necessarily interdependent, our strong preliminary data and expertise in the required methods support the feasibility of the proposed approach. For all experiments, we will analyze the results using rigorous statistical methods and appropriate controls, and will interpret them in the context of our expertise in the biology of C. neoformans and its host. These innovative studies will be enabled by a close collaboration between a cryptococcal biologist and a neuroimmunologist, a powerful combination that will advance fundamental understanding of a central aspect of fungal pathogenesis. This work will additionally open new areas of investigation of a significant pathogen and develop experimental tools that can be used to explore the biology of other neurotropic pathogens.