Thursday, November 21, 2024
11/21/2024
Project Summary Cryptococcus neoformans is an opportunistic fungal pathogen that is inhaled and subsequently encounters a phagocytic cell in the lung and uses this cell to escape the lungs and disseminate to the central nervous system, where it leads to life-threatening meningitis. Cryptococcal meningitis occurs in approximately 225,000 individuals with AIDS each year, resulting in over 181,000 annual deaths. Currently available antifungal drugs are not very effective, they are highly toxic to the host, and the pathogen is developing resistance against these drugs. There is an urgent need to develop new antifungal drugs for cryptococcal infection. Our previous studies showed that individual dendritic cell (DC) lysosomal proteins nostrin, human neutrophil elastase (HNE), matrix metalloprotein 25 (MMP25), coronin, and myeloperoxidase (MPO) kill C. neoformans and are not toxic to mammalian cells. By identifying the mechanism(s) of action and therapeutic potential of these compounds, we can begin to develop novel therapies that can address this public health problem. In fact, therapies that target the fungal pathogen in the lung before it disseminates to the brain would be critical in preventing dissemination – currently no such therapies exist. We hypothesize that these compounds kill C. neoformans by causing fungal cell wall and membrane damage, leading to lysis of the organism and that these compounds will be effective at treating the lung stage of cryptococcal disease. To test this, we will first identify physical attributes of damage to the fungal cells following treatment (misshapen cells, holes in the membrane and cell wall, etc). We will then conduct experiments to determine mutations in fungal genes required for resistance. We will do this by screening C. neoformans mutant libraries and by conducting studies to identify spontaneous suppressor mutants following treatment. We will then test the compounds for antifungal activity in vitro in cell culture before verifying their lack of toxicity in a mouse model. Finally, we will test the efficacy of the antifungal DC lysosome proteins in vivo in a mouse model of pulmonary cryptococcal disease. These proposed studies will offer many opportunities for training undergraduate and graduate students in the laboratory. We will first identify physical effects on C. neoformans following treatment with DC lysosomal components (Aim1). Next, we will identify the mechanisms of antifungal activity by DC lysosome compounds (Aim 2). Finally, we will test the efficacy of novel antifungal compounds in cell culture and in a murine model of pulmonary cryptococcosis (Aim 3).
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