Antifungal mechanisms of action by dendritic cell lysosome proteins

Cryptococcus neoformans is an opportunistic fungal pathogen that primarily affects immunocompromised individuals and can cause cryptococcal meningitis. Approximately 152,000 cases of cryptococcal meningitis occur annually in HIV/AIDS patients, leading to 112,000 yearly deaths. Very few antifungal drugs are available to treat cryptococcal infection, and those are ineffective or toxic. In addition, fungal pathogens such as C. neoformans have become resistant to many types of treatment. Previous studies in our lab showed that proteins from the dendritic cell (DC) lysosome have antifungal activity against C. neoformans, including NOSTRIN, human neutrophil elastase (HNE), matrix metalloproteinase 25 (MMP25), myeloperoxidase (MPO), and coronin. In this study, we tested the antifungal activity of the compound NOSTRIN against the C. neoformans strain H99 using different concentrations of the drug. First, we calculated the minimum inhibitory concentration (MIC). Then, fluorescence microscopy was used to examine structural changes in C. neoformans post-treatment, and mutant libraries were utilized to find genes that exhibited resistance or susceptibility to NOSTRIN. Results showed that NOSTRIN had a similar MIC to the antifungal drug Amphotericin B, with NOSTRIN around 0.25 ug/ml and Amphotericin B around 2 ug/ml. Fluorescence microscopy showed that fungal cells treated with NOSTRIN are releasing intracellular contents following incubation, indicating damage to the fungal cell wall or membrane. Mutant library analysis showed that the deletion of genes such as INM101 increased the percent of surviving mutant cells, while the deletion of NDX2 or PPT1 decreases the percent of surviving mutants. In future studies, we will continue to examine NOSTRIN and other antifungal DC lysosome proteins utilizing microscopy and cryptococcal mutant libraries in order to identify their mechanism(s) of antifungal activity. By researching the presented topics, we hope to gain a better understanding of the effects that these proteins have on C. neoformans, which could potentially lead to more effective therapies for cryptococcosis.