Echinocandins are the newest class of antifungal drugs in clinical use. These agents inhibit β-glucan synthase, which catalyzes the synthesis of β-glucan, an essential component of the fungal cell wall, and have a high clinical efficacy and low toxicity. Echinocandin resistance is largely due to mutations in the gene encoding β-glucan synthase, but the mode of action is not fully understood. We developed fluorescent probes based on caspofungin, the first clinically approved echinocandin, and studied their cellular biology in Candida species, the most common cause of human fungal infections worldwide. Fluorescently labeled caspofungin probes, like the unlabeled drug, were most effective against metabolically active cells. The probes rapidly accumulated in Candida vacuoles, as shown by colocalization with vacuolar proteins and vacuole-specific stains. The uptake of fluorescent caspofungin is facilitated by endocytosis: The labeled drug formed vesicles similar to fluorescently labeled endocytic vesicles, the vacuolar accumulation of fluorescent caspofungin was energy-dependent, and inhibitors of endocytosis reduced its uptake. In a panel comprised of isogenic Candida strains carrying different β-glucan synthase mutations as well as clinical isolates, resistance correlated with increased fluorescent drug uptake into vacuoles. Fluorescent drug uptake also associated with elevated levels of chitin, a sugar polymer that increases cell-wall rigidity. Monitoring the intracellular uptake of fluorescent caspofungin provides a rapid and simple assay that can enable the prediction of echinocandin resistance, which is useful for research applications as well as for selecting the appropriate drugs for treatments of invasive fungal infections.

中文翻译：

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荧光标记的抗真菌药卡泊芬净的高液泡摄取量可预测病原酵母中的棘孢菌素耐药性

Echinocandins是临床上使用的最新一类抗真菌药物。这些药剂抑制β-葡聚糖合酶，其催化真菌细胞壁的必需成分β-葡聚糖的合成，并且具有很高的临床疗效和低毒性。Echinocandin的耐药性在很大程度上是由于编码β-葡聚糖合酶的基因发生突变，但其作用方式尚不完全清楚。我们开发了基于卡泊芬净的荧光探针，卡泊芬净是首个临床批准的棘球菌素，并研究了念珠菌中的细胞生物学，念珠菌是全球人类真菌感染的最常见原因。荧光标记的卡泊芬净探针与未标记的药物一样，对代谢活性细胞最有效。探针在念珠菌中迅速积累空泡，与液泡蛋白和液泡特异性染色剂共定位显示。内吞作用促进了荧光卡泊芬净的摄取：被标记的药物形成类似于荧光标记的内吞囊泡的囊泡，荧光卡泊芬净的液泡积累是能量依赖性的，内吞作用的抑制剂降低了其摄取。由等基因念珠菌组成的小组携带不同β-葡聚糖合酶突变的菌株以及临床分离株，其耐药性与液泡中吸收的荧光药物增加有关。荧光药物的吸收还与几丁质的水平有关，几丁质是一种糖聚合物，可增加细胞壁的硬度。监测细胞内荧光卡泊芬净的吸收提供了一种快速简单的测定方法，可以预测棘球菌素的耐药性，这对于研究应用以及选择合适的药物来治疗侵袭性真菌感染都非常有用。