Leveraging genomic approaches to define sterol transport in Cryptococcus neoformans

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ABSTRACT Cryptococcus neoformans is a devastating opportunistic fungus that causes hundreds of thousands of deaths each year, mainly in developing countries. The most abundant component of its plasma membrane is ergosterol, which is also a compelling topic of study because it is the target of the most powerful antifungal drugs available. How this compound is transported within the cell has never been studied in C. neoformans, constituting a major gap in our knowledge of a critical lipid. Our preliminary results suggest that novel factors are involved in this movement, and that identifying them may be amenable to genetic approaches. The long-term goal of our research is to define the mechanisms of lipid transport in C. neoformans, to advance our fundamental understanding and improve the outcome of this dangerous infection. In this R21 proposal we focus on ergosterol transport. In Aim 1 we will apply a whole genome-scale genetic screen to discover genes whose products influence ergosterol levels at the plasma membrane. In Aim 2 we will isolate and investigate sequences that suppress a defect in retrograde traffic of ergosterol from the plasma membrane to the endoplas- mic reticulum. In Aim 3 we will address the functions of prioritized genes from the first two aims, including studies of growth, virulence, sterol content, and protein and lipid localization. These experiments will be enabled by our expertise in cryptococcal biology and advice from expert colleagues, which we will integrate into thoughtful and rigorous studies. Together, they will increase our understanding of cryptococcal ergosterol transport and position us for further mechanistic investigations of proteins that we discover to be involved in this process. Current antifungal drugs that target ergosterol and its synthesis are hampered by factors that include toxicity, drug interactions, and resistance. It is therefore critical to find other vulnerabilities in this system that can be productively exploited – our proposed studies are a focused step on this path. Completing them will advance genomic methods for fungal discovery, deepen our knowledge of lipid transport, and open the door to future mechanistic studies.