Antifungal Immunity and the Mechanism of Fungal Programmed Cell Death

PROJECT SUMMARYHumans inhale fungal conidia (i.e, vegetative spores) on a daily basis. The ability of the respiratory innateimmune system to prevent germination of inhaled conidia into tissue-invasive hyphae represents a criticalimmunologic checkpoint. Using Aspergillus fumigatus, the most common etiologic agent of invasiveaspergillosis, as a model system for human fungal pathogens, we discovered that conidia undergoprogrammed cell death with apoptosis-like features during interactions with innate immune cells. This findingwas facilitated by a novel fluorescent reporter of fungal physiology that enables visualization and quantitationof fungal apoptosis markers, including histone degradation, caspase activation, and DNA fragmentation.Our work demonstrates that A. fumigatus conidia express an essential and druggable anti-apoptotic protein,termed Bir1, that counters host induction of apoptosis-like programmed cell death by the action of phagocyteNADPH oxidase. Genetic and pharmacologic studies demonstrate that Bir1 expression and activity underlieconidial susceptibility to host apoptosis-like programmed cell death, and in turn, host susceptibility to invasiveaspergillosis. These findings indicate that mammalian fungal immune surveillance exploits a fungal apoptosis-like programmed cell death pathway to maintain barrier immunity in the lung.In this collaborative proposal with two co-investigators, we seek to determine the mechanism through whichBir1 regulates anti-apoptotic activity during fungal-host cell encounters. Our preliminary data support a modelin which Bir1 exerts anti-apoptotic activity via two conserved BIR domains, underlies post-translationalregulation in response to pro-apoptotic stress, regulates candidate fungal caspase-like enzymes as apoptosiseffectors, and demonstrates functional conservation across human pathogenic fungi. Based on theseobservations, our model predicts that fungal apoptosis-like programmed cell death is a general feature offungal-host cell encounters and central to the establishment of invasive fungal disease. We explore this modelin the following aims: (1) define the functional domains and post-translational regulation of Bir1 critical forresistance to host induction of apoptosis-like programmed cell death, (2) define the mechanism of Bir1-mediated resistance to host induction of apoptosis-like programmed cell death, with an emphasis on regulationof a candidate fungal caspase-like activity, and (3) define the role of apoptosis-like programmed cell death andBir1 homologs following Aspergillus nidulans and Candida albicans challenge. The proposed studies aresignificant and innovative because they identify a novel mechanism of immune surveillance and demonstratethat higher eukaryotes can exploit programmed cell death in lower eukaryotes for the purpose of sterilizingimmunity. This work will provide a mechanistic understanding of Bir1 function in regulating host-fungalencounters. Knowledge gained from these studies will inform strategies that target fungal Bir1 homologs andexploit fungal apoptosis-like programmed cell death for therapeutic gain.