Identification and characterization of a Cryptococcus neoformans ATP binding cassette (ABC) transporter-encoding gene, CnAFR1, involved in the resistance to fluconazole
Identification and characterization of a Cryptococcus neoformans ATP binding cassette (ABC) transporter-encoding gene, CnAFR1, involved in the resistance to fluconazole
复制标题
DOI：
10.1046/j.1365-2958.2003.03281.x
复制
发表时间：
2003-01-01
影响因子：
3.6
通讯作者：
Fadda, G
中科院分区：
文献类型：
--
作者：
Posteraro, B;Sanguinetti, M;Fadda, G
隐球菌性脑膜炎是隐球菌病最常见的危及生命的表现，在长时间的抑制性药物治疗过程中，氟康唑耐药是一个可能的事件。在分子水平上了解这种耐药性对于隐球菌病的管理是重要的。为了鉴定新型隐球菌唑类耐药相关基因，选择cDNA消减文库技术作为策略。首先，通过体外暴露于药物，从敏感的临床分离物BPY 22获得氟康唑抗性突变体BPY 22.17。然后，使用消减杂交程序来比较所获得的菌株之间的基因表达。我们鉴定了在氟康唑抗性菌株BPY 22.17中过表达的cDNA，该cDNA用作探针以分离C. neoformans基因组文库。该基因的序列分析鉴定了一个ATP结合盒（ABC）转运蛋白编码基因，称为C。新生儿抗真菌耐药性1（CnAFR 1）。CnAFR 1基因在耐药菌株（BPY 22. 17）中的缺失导致cnafr1对氟康唑的敏感性增强，而在cnafr1中重新引入该基因导致耐药表型恢复，从而证实CnAFR 1参与了氟康唑耐药。新人类因此，我们的研究结果表明，一个积极的药物外排机制，可以参与发展唑类耐药在这个重要的人类病原体。
Resistance to fluconazole is a possible event during prolonged suppressive drug therapy for cryptococcal meningitis, the most frequently encountered life-threatening manifestation of cryptococcosis. The knowledge of this resistance at the molecular level is important for management of cryptococcosis. In order to identify genes involved in azole resistance in Cryptococcus neoformans, a cDNA subtraction library technique was chosen as a strategy. First, a fluconazole-resistant mutant BPY22.17 was obtained from a susceptible clinical isolate BPY22 by in vitro exposure to the drug. Then, a subtractive hybridization procedure was used to compare gene expression between the obtained strains. We identified a cDNA overexpressed in the fluconazole-resistant strain BPY22.17 that was used as a probe to isolate the entire gene in a C. neoformans genomic library. Sequence analysis of this gene identified an ATP Binding Cassette (ABC) transporter-encoding gene called C. neoformans AntiFungal Resistance 1 (CnAFR1). Disruption of CnAFR1 gene in the resistant isolate (BPY22.17) resulted in an enhanced susceptibility of the knock-out mutant cnafr1 against fluconazole, whereas reintroduction of the gene in cnafr1 resulted in restoration of the resistance phenotype, thus confirming that CnAFR1 is involved in fluconazole resistance of C. neoformans. Our findings therefore reveal that an active drug efflux mechanism can be involved in the development of azole resistance in this important human pathogen.