Making and Breaking the Cell Wall of the Pathogenic Fungus Cryptococcus neoformans - PROJECT SUMMARY Cryptococcus neoformans (C. neoformans), is an opportunistic pathogen that causes a fatal meningoencephalitis, claiming about 150,000 deaths annually. Upon infection this yeast rapidly adapts to the host environment, relying heavily on a dramatic expansion of its protective cell wall. Fungal cell walls are highly adaptable in part due to the versatility of the glycans that constitute them, but how these glycans are produced and maintained is not fully understood. This gap needs to be addressed urgently, as one of the three main classes of antifungals, echinocandins (ECs), targets the cell wall in other fungi yet is completely ineffective against C. neoformans. This project's broad, long-term goal is to define the unique glycobiology that allows Cryptococcus to be a successful pathogen. In particular, I will investigate Glucan Organizing Enzyme 1 (Goe1), a glycosyltransferase unique to C. neoformans which has curious involvement in multiple glucan synthesis pathways. We recently reported that cells lacking the corresponding gene (goe1∆ cells) have reduced content of two cell wall polymers, ⍺-1,4- and β-1,3-glucan. The latter is particularly important, since β-1,3-glucan is produced by the target of ECs, a synthase called Fks1. Intriguingly, goe1∆ mutants are extremely sensitive to ECs, which led me to hypothesize that the mechanism of Goe1 may involve Fks1. My preliminary yeast two- hybrid results support this hypothesis by indicating interaction between Goe1 and Fks1. This potential interaction is highly relevant to understanding both cryptococcal biology and echinocandin resistance. In Aim 1, I propose to investigate the role(s) of Goe1 by interrogating Goe1 protein interactions and their functional outcomes. To do this I will first use co-immunoprecipitation to validate Fks1-Goe1 interactions. Aided by structural modeling, I will then predict and perturb residues that mediate interactions and assess the impacts of these perturbations on cellular glycans, Fks1 enzymatic activity, and fungal drug sensitivity. In Aim 2, I propose molecular and genetic approaches to define Goe1's genetic network. I will use RNA-sequencing to determine the transcriptomic response to loss of Goe1and indicate which proteins may work with Goe1 to promote cell wall integrity. In parallel, I will perform a suppressor screen to identify spontaneous genetic changes that arise when goe1∆ mutants are exposed to EC and overcome their susceptibility to these conditions. I will identify and evaluate sequence changes in validated suppressors, which should reveal genes with functions similar to or compensatory for those of Goe1. Understanding the roles of Goe1–and how to inhibit them–can be trailblazing for improving the efficacy of ECs. This research will take place in a highly collaborative and supportive environment, equipped with everything required for the completion of the proposed experiments. It will be supported by a comprehensive training plan that covers avenues for my scientific and professional development in accordance with my long-term career goals.
