Anti‐oxidants reverse DNA damage caused by fluconazole in the pathogenic fungus Cryptococcus neoformans

Abstract

Understanding development of resistance to anti‐fungal drugs in the pathogenic fungus, Cryptococcus neoformans, is crucial for improving anticryptococcal therapy. We have previously determined that presence of anti‐oxidants in growth medium can reverse the fungal inhibition caused by the azole drug, fluconazole. However, the host/pathogen response to co‐treatment with fluconazole and anti‐oxidants remains poorly characterized. In our study, we have used growth assays, infection assays using a heterologous host model, Minimum Inhibitory Concentration (MIC) assays, and flow cytometry to determine how anti‐oxidants influence susceptibility and resistance of C. neoformans to fluconazole. The larval host, Galleria mellonella, was susceptible to C. neoformans and showed higher survival rate when co‐treated with fluconazole and the pathogen. Co‐treatment with select anti‐oxidants, including Pyrrolidine Dithio Carbamate (PDTC), reversed the therapeutic effect of fluconazole. Fluconazole alone causes an increase in DNA content in C. neoformans, detected as additional DNA peaks using flow cytometry, while co‐treatment with fluconazole and select anti‐oxidants reversed the presence of extra stained DNA. Finally, preliminary evidence suggested that C. neoformans lacking the cell cycle regulator, a homologue of Wee1, exhibit higher resistance to fluconazole. Defects in cell division partly account for inhibition of growth of C. neoformans by fluconazole and the ability of anti‐oxidants to reverse an increase in DNA content caused by fluconazole. Our studies suggest that the cell cycle regulator, Wee1 may be important for C. neoformans susceptibility to fluconazole.