Cryptococcus neoformans (CN) is an important human fungal pathogen responsible for thousands of deaths each year, primarily in immunosuppressed individuals. CN makes melanin, a pigment that performs a variety of functions in the plant and animal kingdoms. In fungi, melanin reinforces cell walls, shields against ultraviolet radiation and toxic metals, harnesses high-energy electromagnetic radiation, and contributes to virulence. In addition to contributing to virulence, melanization reduces the susceptibility of fungal cells to antifungal agents and can contribute to the difficulty in treating fungal infections, which are often chronic and notoriously difficult to eradicate. Despite its importance, little is known about the structure of melanin because it is insoluble and amorphous, making it difficult to analyze. This research program takes a multidisciplinary approach to studying the problem of cryptococcal melanization, combining biochemical, cell biology, and spectroscopic (solid state NMR) techniques to uncover the mechanisms of melanization and its impact on the host-microbe interaction. The current application proposes to elucidate the vesicular pathway used to export melanin to the exterior of the cell, investigate the role of lipids in melanin synthesis, investigate how melanin affects the interactions between CN and macrophages, and identify small molecule inhibitors of melanogenesis. Agents that target melanin are potentially valuable because they could be applied against a broad array of pathogenic fungi. Four related but independent aims are proposed: Aim 1. To establish the mechanism for CN cell wall melanization; Aim 2. To determine how neutral lipids in lipid droplets influence melanin synthesis and deposition; Aim 3. To establish the mechanism for how melanization subverts the CN-macrophage interaction; and Aim 4. To identify CN melanin inhibitors and establish their mechanisms of action.