Novel FKS1 and FKS2 modifications in a high-level echinocandin resistant clinical isolate of Candida glabrata
Novel FKS1 and FKS2 modifications in a high-level echinocandin resistant clinical isolate of Candida glabrata
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DOI：
10.1080/22221751.2019.1684209
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发表时间：
2019-01-01
影响因子：
13.2
通讯作者：
Zhao, Yanan
中科院分区：
文献类型：
--
作者：
Hou, Xin;Healey, Kelley R.;Zhao, Yanan
光滑念珠菌的棘白菌素耐药性是一个严重的临床挑战。对棘白菌素泛耐药念珠菌的耐药机制进行了研究。光滑芽孢杆菌（glabrata）分离株（L74株）。通过Alt-R CRISPR-Cas9系统（Fks 1 WT/Fks 2-E655 K，菌株CRISPR 31）和定点突变（菌株fks 1 Delta/Fks 2-E655 K）重建了携带L74中发现的特定突变的FKS突变体。L74株的序列分析显示FKS 1的提前终止密码子W508 stop和FKS 2的热点1区之前的E655 K突变。在ATCC 2001（菌株CRISPR 31）中引入Fks 2-E655 K突变可适度降低敏感性。然而，在fks 1 Delta背景（菌株fks 1 Delta/Fks 2-E655 K）中的相同FKS 2突变导致对棘白菌素的高水平抗性。相对于ATCC 2001，从L74分离的葡聚糖合酶对米卡芬净（MCF）的敏感性显著降低。与ATCC 2001和CRISPR 31相比，L74和fks 1 Delta/Fks 2-E655 K中的FKS 1/FKS 2转录物比率和Fks 1/Fks 2蛋白比率均显著更低（P
Echinocandin resistance in Candida glabrata poses a serious clinical challenge. The underlying resistance mechanism of a pan-echinocandin-resistant C. glabrata isolate (strain L74) was investigated in this study. FKS mutants carrying specific mutations found in L74 were reconstructed by the Alt-R CRISPR-Cas9 system (Fks1 WT/Fks2-E655K, strain CRISPR 31) and site-directed mutagenesis (strain fks1 Delta/Fks2-E655K). Sequence analysis of strain L74 revealed a premature stop codon W508stop in FKS1 and an E655K mutation preceding the hotspot 1 region in FKS2. Introduction of the Fks2-E655K mutation in ATCC 2001 (strain CRISPR 31) conferred a modest reduction in susceptibility. However, the same FKS2 mutation in the fks1 Delta background (strain fks1 Delta/Fks2-E655K) resulted in high levels of resistance to echinocandins. Glucan synthase isolated from L74 was dramatically less sensitive to micafungin (MCF) relative to ATCC 2001. Both FKS1/FKS2 transcript ratios and Fks1/Fks2 protein ratios were significantly lower in L74 and fks1 Delta/Fks2-E655K compared to ATCC 2001 and CRISPR 31 (P