Inhibition of Hsp90 regulates fluconazole resistance in the pathogenic fungus Cryptococcus neoformans

Cryptococcus neoformans is an encapsulated yeast, found in the lungs of immunocompromised hosts. In the human host, Cryptococcusis subject to multiple forms of environmental stress, including heat stress. Heat shock protein 90 (Hsp90) is an essential protein that has been shown to protect fungal cells against extreme temperature in the human host and to assist in proper cell division. Hsp90 has also been implicated in resistance to the anti-fungal drug fluconazole. To better characterize the role of Hsp90 in fluconazole resistance in Cryptococcuswe addressed two questions: 1. Does concomitant partial inhibition of Hsp90 and treatment with fluconazole lead to increased sensitivity to fluconazole and 2. Does prolonged partial inhibition of Hsp90 prior to treatment with fluconazole lead to subsequent increase in development of fluconazole resistance. Using growth assays, disk assays, and a standardized e-test to determine the Minimum Inhibitory Concentration (MIC) of fluconazole, we found that indeed, pharmacological partial inhibition of Hsp90 with radicicol, concomitant with fluconazole treatment leads to increased sensitivity to fluconazole. However, initial pre-treatment of Cryptococcuswith sub-inhibitory concentration of radicicol followed by exposure to fluconazole leads to development of fluconazole resistant Cryptococcuscolonies. We characterized the resistant colonies, which show a lower MIC to fluconazole compared to cells that were never exposed to radicicol and fluconazole. Cells derived from the resistant colonies exhibit slower growth on drug-free media, and lose resistance to the drug after passaging in drug-free media. Based on the fact that resistance to fluconazole is lost when fluconazole is removed from the environment, we hypothesize that compromising Hsp90 function during the pre-treatment with radicicol triggers aneuploidy in Cryptococcus neoformans. Furthermore, we hypothesize that the resistant colonies are aneuploids that possess an altered chromosome number to maintain drug resistance in response to an environmental insult, such as presence of fluconazole.