Mechanisms of immune evasion by a neuroinvasive fungal pathogen

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Project Summary Cryptococcus neoformans is a neuroinvasive yeast that causes fatal meningitis in immunocompromised individuals, resulting in >180,000 annual deaths representing 15% of global AIDS-related mortality. Cryptococci initiate infection in the lungs, where they must overcome host innate immunity to establish a replicative niche and disseminate to the central nervous system. Dectin-1 is a critical pattern recognition receptor (PRR) that initiates antifungal defenses in response to β-glucans, a pathogen-associated molecular pattern (PAMP) ubiquitous in fungal cell walls. While Dectin-1 is essential for defense against important pathogens such as Candida albicans and Aspergillus fumigatus, this receptor fails induce protective responses during C. neoformans infection, despite the presence of β-glucans in the cryptococcal cell wall. This suggests that cryptococci employ yet-undiscovered mechanisms to evade Dectin-1 sensing. The objective of this proposal is to define these mechanisms. To accomplish this, I will test the central hypothesis that C. neoformans evades Dectin-1 sensing using virulence factors that shield PAMPs and/or suppress host inflammatory signaling. Specifically, I aim to 1) identify fungal genes necessary for Dectin-1 evasion and 2) determine the relative contributions of PAMP shielding and inflammatory suppression to this process. Preliminary work in the Madhani lab has produced a library of >4000 well-validated, single-gene C. neoformans deletion strains, and I have developed a robust sequencing-based workflow to quantify these mutants in complex pools. Leveraging these tools, I will systematically identify fungal virulence factors required for Dectin-1 evasion using an in vivo murine pulmonary infection model and an in vitro macrophage stimulation model. To then define mechanisms of Dectin- 1 evasion, I will profile Dectin-1 evasion mutants for diverse phenotypes diagnostic of roles in either PAMP shielding or inflammatory suppression. This will classify novel evasion factors as acting through one or both of these mechanisms, thereby revealing the extent to which PAMP shielding and inflammatory suppression influence Dectin-1 evasion. Thus, by identifying the factors that subvert a key antifungal sensing pathway and defining the mechanisms through which this pathway is neutralized, this work addresses an important knowledge gap in cryptococcal pathogenesis and will provide a foundational understanding for how invasive fungi overcome innate defenses.