Abstract Cryptococcal meningitis is an AIDS-defining condition and it is responsible for 15% of the deaths in AIDS patients. The disease has mortality rates up to 70% and it claims hundreds of thousands of lives each year. The existing antifungal drugs are not always effective and there is no vaccine available against cryptococcosis. The challenges of preventing and treating this disease motivate us to investigate cryptococcal pathogenesis and identify cryptococcal pathways that can induce a protective host response. Cryptococcus neoformans can undergo yeast-to-filament morphological transition. We and others have shown that morphotype has a profound effect on cryptococcal interaction with various hosts. In mammalian models of cryptococcosis, the yeast form is pathogenic while the filamentous form is attenuated/abolished in virulence. Previously, we identified a key regulator of filamentation, Znf2. Deletion of ZNF2 locks cells in the yeast form and enhances virulence. Overexpression of ZNF2 promotes filamentation in vitro and in vivo, abolishes virulence, and can offer rare sterilizing immunity against an otherwise lethal challenge. Based on these studies, we hypothesize that activating the cryptococcal filamentation program can elicit protective host responses and alleviate cryptococcosis. The goal of this application is to characterize filamentation pathways and identify those that induce protective host responses and attenuate cryptococcal virulence. Capitalizing on our recent discoveries of self-filamentation in natural isolates of C. neoformans species complex (serotype A and D), we performed multiple large genetic screens and identified candidate genes that play important roles in filamentation in vitro. Among these candidates, multiple components of the osmotic sensing pathway suppress filamentation in the serotype A reference strain H99 by inhibiting nuclear translocation of the transcription factor Crz1, which acts upstream of Znf2. In Aim 1, we will define the mechanism by which Crz1 distinguishes different stimuli to activate filamentation. In Aim 2, we will characterize the identified candidates and establish their genetic relationship with Znf2 in controlling cryptococcal virulence and morphology in vivo. In Aim 3, we will test the avirulent strains for their immunization effect and their combined effect with Znf2 on host immunity. The work will generate a set of Cryptococcus morphological mutants that induce protective host responses. These findings will reveal targets that can be exploited in the future by us and others to investigate new measures against this deadly fungal disease.