Deciphering fungal pathogen-specific cell cycle mechanisms to fuel drug discovery - Project Summary Fungal infections are a pressing and underappreciated threat to global public health, causing an estimated 2.5 million deaths each year. One major contributing agent is Cryptococcus neoformans, a globally distributed opportunistic fungal pathogen capable of causing life-threatening meningitis in immunocompromised individuals. Available treatment options for cryptococcal infections and fungal infections in general are limited, underscoring the urgent need for new antifungal development. Two main challenges in combating fungal pathogens are the cellular similarities between fungi and human hosts and their abilities to evade the host immune system. In the case of C. neoformans, the typical small haploid yeast cells undergo a striking morphological transition to form giant, highly polyploid “titan cells” to escape phagocytosis by host immune cells. A key to C. neoformans’s ability to proliferate and form titan cells in the host is the cell cycle machinery. Despite being essential for pathogen proliferation and pathogenicity, the cell cycle machinery has been traditionally excluded as a target for antifungal drug development due to its high conservation between fungi and humans. However, there are differences and modifications in the cell cycle program that are fungal-specific or unique to C. neoformans. Here, I propose to exploit these cell cycle variations essential for fungal proliferation or immune evasion for antifungal drug development. To realize this vision, I have identified two promising cell cycle variations to investigate in C. neoformans. Aim 1 will focus on a fungal-specific cell cycle pathway known as the mitotic exit network (MEN). The MEN is highly conserved in fungi and functions in a fungal-specific cell cycle checkpoint in the budding yeast Saccharomyces cerevisiae, thus providing a potential target for broad-spectrum antifungals. This proposal will determine the function and signaling mechanisms for the MEN in C. neoformans. In addition, we will develop a strategy to perform high throughput genetic interaction mapping using CRISPR/Cas9-mediated gene editing and an inducible degron system to dissect the regulatory network surrounding the MEN in C. neoformans. The goal of Aim 2 is to pinpoint the specific cell cycle alterations that program the reversible titan cell formation. In addition to promoting immune evasion via polyploidization, the titan cells undergo an unusual reductive division to produce haploid daughters to facilitate dissemination to distant tissues such as the brain. We will systematically track and perturb cell cycle progression during titan cell formation and division to distinguish potential models for the polyploidization and reductive division. In parallel, we will perform high throughput phenotyping with a genome-wide depletion library to identify key regulators for the cell cycle alternations in both processes. The proposed study will help identify new drug targets for the important human fungal pathogen C. neoformans and provide a proof of principle for the unconventional and generalizable approach of targeting the cell cycle for antifungal drug development.
