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 fks1Δ/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 fks1Δ background (strain fks1Δ/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 fks1Δ/Fks2-E655K compared to ATCC 2001 and CRISPR 31 (P <0.05). Mice challenged with CRISPR 31 and fks1Δ/Fks2-E655K mutants failed to respond to MCF. In conclusion, the high-level of echinocandin resistance in the clinical isolate of C. glabrata L74 was concluded to result from the combination of null function of Fks1 and the point mutation E655K in Fks2.

中文翻译：

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新型的FKS1和FKS2修饰在光滑念珠菌的高棘皮菌素抗性临床分离株中。

光滑念珠菌对棘球chin碱的耐药性构成了严重的临床挑战。在这项研究中，研究了对泛棘皮菌素抗性的光滑念珠菌分离株（菌株L74）的潜在抗性机理。通过Alt-R CRISPR-Cas9系统（Fks1 WT / Fks2-E655K，CRISPR 31株）和定点诱变（菌株fks1Δ/ Fks2-E655K）重建了在L74中发现的携带特定突变的FKS突变体。菌株L74的序列分析显示，FKS1中的密码子W508stop提前终止，而FKS2热点1区域之前的E655K突变。在ATCC 2001中引入Fks2-E655K突变（CRISPR 31株）使其敏感性适度降低。但是，在fks1Δ背景中相同的FKS2突变（菌株fks1Δ/ Fks2-E655K）导致对棘手and蛋白的高水平抗性。与ATCC 2001和CRISPR 31相比，从L74分离出的葡聚糖合酶相对于米卡芬净（MCF）对米卡芬净（MCF）的敏感性大大降低.L74和fks1Δ/ Fks2-E655K的FKS1 / FKS2转录本比率和Fks1 / Fks2蛋白比率均显着较低。 P <0.05）。用CRISPR 31和fks1Δ/ Fks2-E655K突变体攻击的小鼠对MCF无效。结论是，临床上分离到的光滑念珠菌L74中的棘皮菌素耐药性较高，是由于Fks1的无效功能和Fks2中的点突变E655K的结合所致。用CRISPR 31和fks1Δ/ Fks2-E655K突变体攻击的小鼠对MCF无效。结论是，临床上分离到的光滑念珠菌L74中的棘皮菌素耐药性较高，是由于Fks1的无效功能和Fks2中的点突变E655K的结合所致。用CRISPR 31和fks1Δ/ Fks2-E655K突变体攻击的小鼠对MCF无效。结论是，临床上分离到的光滑念珠菌L74中的棘皮菌素耐药性较高，是由于Fks1的无效功能和Fks2中的点突变E655K的结合所致。