ABSTRACT Cryptococcus neoformans (Cn) is the most common cause of fungal meningoencephalitis, a life-threatening disease. Despite extensive efforts, the mechanism whereby Cn crosses the blood-brain barrier (BBB) and causes meningitis remains poorly understood. Our previous study discovered that inositol, a host sugar metabolite highly abundant in the brain, promotes fungal traversal of the BBB and plays a critical role in host- pathogen interactions during infection of the central nervous system (CNS). In our previous funding period, we have demonstrated that Cn contains an unusually complex inositol uptake system that is distinct from those characterized in other fungi. Thus, Cn may be uniquely adapted to thrive in the inositol-rich environment of the CNS and to utilize inositol-dependent pathways for pathogenesis. Our data have confirmed that (1) growth of Cn under inositol-rich conditions enhanced virulence in the murine model, (2) fungal cells deficient in inositol uptake and catabolism were also deficient in virulence during CNS infection, (3) inositol induced production of fungal capsule, a major virulence factor, and hyaluronic acid, a cell surface molecule involved in fungal binding to the BBB, and (4) inositol induced a unique capsule structure enriched in a specific mannosylated structure group that helps the fungus evade the host immune response. We also identified an inositol receptor Itr4 and a transcription factor Hlh6 that are required for inositol function in Cn. Our central hypothesis is that cryptococcal cells sense and utilize host inositol to modify the fungal cell surface in a way that promotes penetration of the BBB and development of cryptococcal meningoencephalitis. The overarching goal of this proposal is to understand how this pathogen senses and utilizes host nutrient inositol to establish human brain infections. To further test our hypothesis, we propose two related but independent Specific Aims: 1) Characterize Inositol mediated fungal cell surface modification in Cryptococcus-host interaction using genetics, molecular biology and biophysical approaches in combination of in vitro BBB system and in vivo animal models. Fungal strains with single motif capsule structure will be utilized for this study. 2) Define the inositol sensing and regulatory mechanism important for fungal pathogenesis by characterizing the inositol sensor Itr4 and inositol transcription factors and the pathways they regulated. Moreover, we will test a number of inhibitors on inositol function for their potential antifungal activity to explore the possibility to develop the fungal inositol function as a potential therapeutic target. The proposed study will provide a mechanistic understanding of inositol utilization in Cn and its effects on fungal pathogenesis, especially during CNS infections.