Mutation of G234 amino acid residue in Candida albicans drug-resistance-related protein Rta2p is associated with fluconazole resistance and dihydrosphingosine transport
Mutation of G234 amino acid residue in Candida albicans drug-resistance-related protein Rta2p is associated with fluconazole resistance and dihydrosphingosine transport
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DOI：
10.1080/21505594.2015.1051296
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发表时间：
2015-07
期刊：
影响因子：
5.2
通讯作者：
Shiqun Zhang;Qi Miao;Liping Li;Lulu Zhang;Lan Yan;Yuchi Jia;Yongbing Cao;Yuan-Ying Jiang
中科院分区：
文献类型：
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作者：
Shiqun Zhang;Qi Miao;Liping Li;Lulu Zhang;Lan Yan;Yuchi Jia;Yongbing Cao;Yuan-Ying Jiang
唑类药物的广泛和重复使用导致白念珠菌耐药性的迅速发展。我们前期的研究发现，Rta 2 p是一种具有7个跨膜结构域的膜蛋白，参与了钙调神经磷酸酶介导的唑类药物耐药和鞘氨醇长链碱基释放。白色念珠菌。对Rta 2 p跨膜结构域中的保守氨基酸进行定点诱变。结果表明，C.采用最低抑菌浓度法和杀灭法检测白念珠菌对氟康唑的体外抑制作用，采用小鼠全身念珠菌感染模型检测氟康唑对白念珠菌的体内治疗作用。通过NBD标记D-邻二氢鞘氨醇的摄取和释放实验检测了二氢鞘氨醇的转运活性，并对鞘脂生物合成抑制剂进行了敏感性测定。我们成功构建了14株Rta 2 p突变株，通过最低抑菌浓度筛选，发现Ca 2+不能完全诱导G158 E和G234 S突变株对氟康唑产生耐药性。此外，我们证实了G234 S突变体增强了氟康唑对系统性念珠菌病的治疗效果，并通过减少其释放而显著增加了二氢鞘氨醇的积累。但G158 E突变体不影响药物的体内疗效和二氢鞘氨醇的转运。白色念珠菌。G234在C.白念珠菌在钙调神经磷酸酶介导的氟康唑耐药性和二氢鞘氨醇转运中至关重要。
Widespread and repeated use of azoles has led to the rapid development of drug resistance in Candida albicans. Our previous study found Rta2p, a membrane protein with 7 transmembrane domains, was involved in calcineurin-mediated azole resistance and sphingoid long-chain base release in C. albicans. Conserved amino acids in the transmembrane domain of Rta2p were subjected to site-directed mutagenesis. The sensitivity of C. albicans to fluconazole in vitro was examined by minimum inhibitory concentration and killing assay, and the therapeutic efficacy of fluconazole in vivo was performed by systemic mice candidiasis model. Furthermore, dihydrosphingosine transport activity was detected by NBD labeled D-erythro-dihydrosphingosine uptake and release assay, and the sensitivity to sphingolipid biosynthesis inhibitors. We successfully constructed 14 mutant strains of Rta2p, screened them by minimum inhibitory concentration and found Ca2+ did not completely induce fluconazole resistance with G158E and G234S mutations. Furthermore, we confirmed that G234S mutant enhanced the therapeutic efficacy of fluconazole against systemic candidiasis and significantly increased the accumulation of dihydrosphingosine by decreasing its release. However, G158E mutant didn't affect drug therapeutic efficacy in vivo and dihydrosphingosine transport in C. albicans. G234 of Rta2p in C. albicans is crucial in calcineurin-mediated fluconazole resistance and dihydrosphingosine transport.