Identification of the antifungal mechanism of EIPE-1 against Cryptococcus neoformans

Cryptococcus neoformans is a fungal pathogen found worldwide in soil and bird droppings that can cause disease in both immunocompromised and immunocompetent hosts. Cryptococcal meningitis has received global attention and is credited with 152,000 cases of cryptococcal meningitis yearly in the HIV/AIDS population, killing approximately 112,000 people each year. Treatment options for fungal infections are limited, many treatments are toxic, and fungal organisms are becoming resistant to current treatments. Therefore, the discovery of novel antifungal therapies is critical for fighting these deadly infections. Previous studies in our lab showed that a eumelanin-inspired compound, EIPE-1, has antifungal activity against C. neoformans. EIPE-1 treatment led to misshapen cells, and transcriptional data showed differential regulation of fungal genes involved in cell wall synthesis, membrane assembly, and cell stress. Therefore, we hypothesized that fungal strains with mutations in these signaling pathways will be resistant to EIPE-1 treatment. For this, we have screened mutants from the C. neoformans mutant library for those that are available for transcription factors, kinases, and phosphatases. Mutants were mixed in a competition with an m-cherry fluorescent cryptococcal strain and treated with the MIC concentration of EIPE-1 or were left untreated as a control. Following a 24h incubation, cells were analyzed on a flow cytometer to evaluate resistant mutants. Data show that the de mutants tested are more susceptible to EIPE-1 treatment than wild-type cryptococcal cells. Additional studies will examine further mutants to uncover those more resistant to EIPE-1 treatment. Future studies will verity these data by incubating the mutant strains with increasing concentrations of EIPE-1, which will verify their importance in resistance to EIPE-1. Finally, we will examine the how EIPE-1 interacts with the signaling pathway(s) where these genes are located by Western blot to examine phosphorylation of proteins in the identified pathways. These studies will enable us to pinpoint the mechanism of action of EIPE-1 against C. neoformans.