ABSTRACT: Fluconazole (FLC), a triazole antifungal drug, is widely used for the maintenance therapy of cryptococcal meningoencephalitis, the most common opportunistic infection in AIDS patients, but this usage predisposes to the appearance of FLC resistance, especially in patients with no or limited access to highly active antiretroviral therapy. We used microarray analysis to examine changes in the gene expression profile of C. neoformans reference strain H99 (serotype A) following exposure to FLC in order to study the adaptive cellular responses to drug stress. Simultaneous analysis of over 6,823 C. neoformans gene transcript levels revealed that 476 genes were responsive to FLC. Up-regulated expression was observed, as expected, for genes involved in the ergosterol biosynthesis, including ERG13, ERG1, ERG7, ERG25, ERG2, ERG3, ERG5, and that encoding the azole target, ERG11, but also for the gene SRE1, that encodes a well-known regulator of sterol homeostasis in C. neoformans. In addition, several genes such as those involved in a wide variety of important cellular processes (i.e., lipid and fatty acid metabolism, cell wall maintenance, stress, virulence, etc.), were found to be up-regulated in response to fluconazole treatment. Some of these genes may represent potential therapeutic targets to be exploited in anticryptococcal therapy. Conversely, expression of AFR1, the major transporter of azoles in C. neoformans, was shown to be not affected by exposure to FLC, thus suggesting a minor involvement in the C. neoformans short-term adaptation to the azole drug. We studied the transient response of C. neoformans to fluconazole by analyzing differences in gene expression prior to and after exposure of strain H99. Three biological replicates were performed for each condition (FLC-exposed and -not exposed (controls)). The cells were exposed to 10 µg of FLC/ml (1/2 x MIC) or distilled water (controls) for one doubling time (90 min).
