Expression Profiling of the Response to Short-Term Exposure to Fluconazole in Cryptococcus neoformans Serotype A

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).

氟康唑(FLC)作为三唑类抗真菌药物，被广泛用于隐球菌性脑膜炎的维持治疗——而隐球菌性脑膜炎是艾滋病患者最常见的机会性感染，但该用药方案易诱发FLC耐药性的产生，尤其在无法获得或仅能有限获得高效抗反转录病毒治疗的患者群体中。本研究采用微阵列分析技术，检测新型隐球菌(Cryptococcus neoformans)参考菌株H99（血清型A）在暴露于FLC后的基因表达谱变化，以探究其对药物应激的适应性细胞应答。对超过6823个新型隐球菌基因转录本水平的同步分析显示，共有476个基因对FLC产生应答。正如预期，麦角固醇生物合成相关基因的表达出现上调，包括ERG13、ERG1、ERG7、ERG25、ERG2、ERG3、ERG5以及编码三唑类药物靶标的ERG11；此外，编码新型隐球菌固醇稳态经典调控因子的SRE1基因表达同样上调。此外，多项参与多种重要细胞过程（如脂质与脂肪酸代谢、细胞壁维持、应激响应、毒力等）的基因，也被发现可在FLC处理后表达上调。其中部分基因有望成为抗隐球菌治疗的潜在药物靶点。与之相反，新型隐球菌主要的三唑类转运蛋白编码基因AFR1的表达并未受FLC暴露的影响，这提示该基因在新型隐球菌短期适应三唑类药物的过程中仅发挥微弱作用。本研究通过分析H99菌株在FLC暴露前后的基因表达差异，探究新型隐球菌对氟康唑的瞬时应答。每组实验条件（FLC暴露组与未暴露对照组）均设置3次生物学重复。将菌株置于10μg/mL FLC（为最低抑菌浓度(Minimum Inhibitory Concentration, MIC)的1/2倍）或蒸馏水（对照组）中培养一个倍增时长（90分钟）。