Fungal infections in the central nervous system (CNS) cause significant morbidity and mortality. Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening meningoencephalitis most commonly in populations with impaired immunity. The number of cases of cryptococcosis worldwide is estimated at 1 million with over 600,000 deaths per year. The mortality associated with cryptococcal meningitis is unacceptably high especially in HIV-infected populations in resource-poor settings. In the United States, a total of 3210 cryptococcosis-related deaths were identified between the years 2000-2010. Cerebral cryptococcosis was the most commonly reported clinical manifestation of the disease. Indeed without rapid intervention, cryptococcal meningoencephalitis is universally fatal regardless of the immune status of the host. Unfortunately, even after successful treatment and recovery neurological sequelae can remain. For C. neoformans to invade the CNS it must first penetrate the blood-brain barrier (BBB) - this is an incredible feat given that the primary role of the BBB is to protect the brain from potentialy harmful agents. C. neoformans crosses the BBB primarily via a transcellular mechanism - this involves attachment to- and internalization by the BBB (on the blood- side), followed by transmigration across the BBB cytoplasm and finally exiting on the abluminal side (the brain side) of the BBB. The molecular mechanisms that govern this complex process are largely unresolved and this has negatively impacted patient outcome. We originally proposed that proteins on the surface of C. neoformans were likely involved during the transmigration of the BBB. Subsequently we were the first to identify and demonstrate that a secreted fungal metalloprotease, MPR1, is required and sufficient for attachment and internalization of C. neoformans by the BBB both in vitro and in vivo. MPR1 belongs to a newly identified class of M36 metalloproteases unique to some fungi whose substrates and interacting protein partners are largely unknown. We found that MPR1 may be specific for the BBB since MPR1 is not required for dissemination or colonization of other organs like lungs, kidneys, spleen or heart. Importantly, MPR1 is sufficient for transmigration because the sole expression of CnMPR1 in Saccharomyces cerevisiae, yeast that cannot normally migrate across the BBB, suddenly gained the ability to do so. Given the central role of Mpr1 in penetrating the BBB, we will test th hypothesis that Mpr1 promotes a permeable BBB by proteolytically activating or unmasking BBB surface receptors, ligands and/or adhesion proteins and thereby facilitates the transcellular migration of C. neoformans. In order to test this prediction two aims will be pursued. In aim 1 we will identify protein targets on the surface of the BBB that mediate attachment and internalization of fungal cells. In aim 2 we will resolve the full degradome of MPR1 via a system-wide substrate discovery platform. The impact of establishing a workable and productive methodology for further exploration of this biologically relevant interface is enormous. Since MPR1 belongs to a novel, poorly characterized class of fungal metalloproteases that are present in other fungi that can have a high rate of CNS involvement, the implications of this study are far reaching. Moreover, this study will advance translational goals of blocking MPR1 activity as a therapeutic tool in preventing fungal meningoencephalitis and in developing an MPR1-based drug delivery platform technology for the treatment of neurological disorders.